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WO2025088147A1 - Theranostic psma-targeting ligands for the diagnosis and treatment of psma-expressing cancers and their solid phase synthesis - Google Patents

Theranostic psma-targeting ligands for the diagnosis and treatment of psma-expressing cancers and their solid phase synthesis Download PDF

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Publication number
WO2025088147A1
WO2025088147A1 PCT/EP2024/080276 EP2024080276W WO2025088147A1 WO 2025088147 A1 WO2025088147 A1 WO 2025088147A1 EP 2024080276 W EP2024080276 W EP 2024080276W WO 2025088147 A1 WO2025088147 A1 WO 2025088147A1
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Prior art keywords
group
fmoc
compound
acid
psma
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Inventor
Martin Schäfer
Ulrike Bauder-Wuest
Mareike ROSCHER
Yvonne REMDE
Martina BENEŠOVÁ-SCHÄFER
Cyril BARINKA
Zsófia KUTILOVÁ
Lucia MOTLOVÁ
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0402—Organic compounds carboxylic acid carriers, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds

Definitions

  • the present invention generally relates to the field of radiopharmaceuticals and their use in nuclear medicine as tracers, imaging agents and radiopharmaceuticals for the treatment of various disease states of PSMA-expressing cancers, especially prostate cancer, and metastases thereof. Further the present invention provides synthetic procedures for the synthesis of such PSMA-targeting ligands,
  • Prostate cancer is the leading cancer in the US and European population in men. At least 1-2 million men in the western hemisphere suffer from prostate cancer and it is estimated that the disease will strike one in six men between the ages of 55 and 85. There are more than 300,000 new cases of prostate cancer diagnosed each year in USA. The mortality from the disease is second only to lung cancer.
  • imaging methods with high resolution of the anatomy such as computed tomography (CT), magnetic resonance (MR) imaging and ultrasound, predominate for clinical imaging of prostate cancer.
  • An estimated annual $ 2 billion is currently spent worldwide on surgical, radiation, drug therapy and minimally invasive treatments.
  • CT computed tomography
  • MR magnetic resonance
  • PCa imaging agents include radiolabeled choline analogs [ 18 F]fluorodihydrotestosterone ([ 18 F]FDHT), anti-l-amino-3-[ 18 F]fluorocyclobutyl-l-carboxylic acid (anti[ 18 F]FACBC), [ n C]acetate and l-(2-deoxy-2-[ 18 F]flouro-L-arabinofuranosyl)-5-methyluracil ([ 18 F]FMAU) (Scher, B.; et al. Eur J Nucl Med Mol Imaging 2007, 34, 45-53; Rinnab, L; et al.
  • tumors may express unique proteins associated with their malignant phenotype or may over-express normal constituent proteins in greater number than normal cells.
  • the expression of distinct proteins on the surface of tumor cells offers the opportunity to diagnose and characterize disease by probing the phenotypic identity and biochemical composition and activity of the tumor.
  • Radioactive molecules that selectively bind to specific tumor cell surface proteins provide an attractive route for imaging and treating tumors under non-invasive conditions.
  • a promising new series of low molecular weight imaging agents targets the prostate-specific membrane antigen (PSMA) (Mease R.C. et al. Clin Cancer Res. 2008, 14, 3036-3043; Foss, C.A.; et al.
  • PSMA prostate-specific membrane antigen
  • PSMA is a trans-membrane, 750 amino acid type II glycoprotein that has abundant and restricted expression on the surface of PCa, particularly in androgen-independent, advanced and metastatic disease (Schulke, N.; et al. Proc Natl Acad Sci U S A 2003, 100, 12590-12595). The latter is important since almost all PCa become androgen independent over the time. PSMA possesses the criteria of a promising target for therapy (Schulke, N.; et al. Proc. Natl. Acad. Sci. U S A 2003, 100, 12590-12595).
  • the PSMA gene is located on the short arm of chromosome 11 and functions both as a folate hydrolase and neuropeptidase.
  • GCPII glutamate carboxypeptidase II
  • brain PSMA glutamate carboxypeptidase II
  • NAG N-acetyl aspartyl glutamate
  • NAA N-acetyl aspartate
  • the radio-immunoconjugate of the anti-PSMA monoclonal antibody (mAb) 7E11, known as the PROSTASCINT® scan, is currently being used to diagnose prostate cancer metastasis and recurrence.
  • this agent tends to produce images that are challenging to interpret (Lange, P.H. PROSTASCINT® scan for staging prostate cancer. Urology 2001 , 57, 402-406; Haseman, M.K.; et al. Cancer Biother Radiopharm 2000, 15, 131-140; Rosenthal, S.A.; et al. Tech Urol 2001 , 7, 27-37).
  • monoclonal antibodies have been developed that bind to the extracellular domain of PSMA and have been radiolabeled and shown to accumulate in PSMA-positive prostate tumor models in animals.
  • diagnosis and tumor detection using monoclonal antibodies has been limited by the low permeability of the monoclonal antibody in solid tumors.
  • radionuclides are known to be useful for radioimaging or cancer radiotherapy, including U 1 ln, 90 Y, 68 Ga, 177 Lu, " m Tc, 123 I and 131 I. Recently it has been shown that some compounds containing a glutamate-urea-glutamate (GUG) or a glutamate-urea-lysine (GUL) recognition element linked to a radionuclide-ligand conjugate exhibit high affinity for PSMA.
  • GAG glutamate-urea-glutamate
  • GUL glutamate-urea-lysine
  • WO 2015/055318 new imaging agents with improved tumor targeting properties and pharmacokinetics were described. These compounds comprise a motif specifically binding to cell membranes of cancerous cells, wherein said motif comprises a prostate-specific membrane antigen (PSMA), that is the above mentioned glutamate-urea-lysine motif.
  • PSMA prostate-specific membrane antigen
  • the preferred molecules described in WO 2015/055318 further comprise a linker which binds via an amide bond to a carboxylic acid group of DOTA as chelator.
  • Some of these compounds have been shown to be promising agents for the specific targeting of prostate tumors. The compounds were labeled with 177 Lu (for therapy purposes) or 68 Ga (for diagnostic purposes) and allow for visualization and targeting of prostate cancer for radiotherapy purposes.
  • MB- 17 (or PSMA-617) was disclosed.
  • the ligand MB-24 (or PSMA-624) disclosed in WO 2015/055318 and comprising an aromatic linker moiety instead of the cyclohexyl moiety present in PSMA-617 demonstrated a high internalization rate. However, it also showed higher retention in non-targeted organs and tissues in comparison with PSMA-617. See also Benesova M, Bauder-Wiist U, Schafer M, Klika KD, Mier W, Haberkorn U, Kopka K, Eder M.
  • PSMA ligands which enable various strategies for the detection, treatment and management of PSMA-expressing cancers, in particular prostate cancer.
  • the synthesis of the binding motif described in the prior art is carried out via a solid phase approach employing hazardous triphosgene, which generally limit the solid phase synthetic approach for PSMA ligands. Furthermore, the method used so far is incompatible with a large number of synthetic building blocks to be used due to solubility problems.
  • the solution of said object is achieved by providing the embodiments characterized in the claims.
  • the inventors found new compounds which are useful and advantageous radiopharmaceuticals and which can be used in nuclear medicine as tracers, imaging agents and for the treatment of various disease states of PSMA-expressing cancers, in particular prostate cancer. These compounds are described in more detail below.
  • the present invention relates to a compound of formula (1) or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 is H or -CH3, preferably H, wherein R 2 , R 3 and R 4 are independently of each other, selected from the group consisting of - CO2H, -SO2H, -SO3H, -OSO3H, -PO2H2, -PO3H2, -OPO3H2, -CONH2 and -C0NH0H,.
  • Q 2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
  • the present invention relates to a complex comprising
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound, as described above or below, or a pharmaceutically acceptable salt or solvate thereof, as described above or below, or a complex, as described above or below.
  • the present invention relates to a compound, as described above or below, or a pharmaceutically acceptable salt or solvate thereof, or a complex, as described above or below, or a pharmaceutical composition as described above or below, for use in treating or preventing PSMA-expressing cancers, in particular prostate cancer, and/or metastases thereof.
  • the present invention relates to a method for preparing a compound of formula (1) or a pharmaceutically acceptable salt or solvate thereof, as described above, the method comprising:
  • the present invention relates to a method for preparing a compound of formula (1) or a pharmaceutically acceptable salt or solvate thereof, as described above, the method comprising:
  • the present invention relates to a method for preparing a resin bound PSMA binding moiety having the structure preferably having the structure as intermediate product for the solid phase synthesis of PSMA ligands, preferably for the synthesis of a PSMA ligand according to embodiment 1 to 11, the method comprising the steps,
  • step (c) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin.
  • n is 1, 2 or 3, more preferably 1.
  • the compound of formula (I) preferably has the structure (la), wherein R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
  • all natural or non-natural amino acids having a chiral C-atoms shall have D- and/or L-configuration; also combinations within one compound shall be possible, i.e. some of the chiral C-atoms may be D- and others may be L-configuration.
  • all amino acid residues present in the compound have L-configuration.
  • XAA 1 preferably has L-configuration, compound (I) thus having the structure (laa) wherein R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
  • the compound of the invention has preferably the structure (lb), more preferably, the structure (Ibb)
  • R 1 is H or -CH3, preferably H.
  • R 2 , R 3 and R 4 are independently of each other, selected from the group consisting of -CO2H, - SO2H, -SO3H, -OSO3H, -PO2H2, -PO3H2, -OPO3H2, -CONH2 and -CONHOH. More preferably, R 2 , R 3 and R 4 are CO2H.
  • carbon atoms present in the urea backbone of the compound if the present invention may be present in any stereoisomeric form, preferably, however, the compounds have the following structures: preferably wherein R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
  • the compound of the invention has the structure (Ib-1), more preferably, the structure (Ibb-1) (lbb-1), wherein R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
  • Q 2 is preferably selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl.
  • alkyl refers to a saturated acyclic hydrocarbon moiety comprising 1 to 10 carbon atoms, preferably 1-6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms, preferably 1 carbon atom.
  • aryl refers to optionally substituted 6- membered aromatic ring, i.e. optionally substituted phenyl group.
  • alkylaryl refers to aryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-aryl-) and which are linked via the alkyl group to the -CH2- group and via the aryl group to the carbonyl group.
  • arylalkyl refers to aryl groups linked via an alkyl group to the carbonyl group and via the aryl group to the -CH2- group (-aryl-alkyl-).
  • heteroaryl (-heteroaryl-), as used in this context of the invention, means optionally substituted, 5- and 6-membered aromatic rings, containing one or more, for example 1 to 4, such as 1, 2, 3, or 4, heteroatoms in the ring system. If more than one heteroatom is present in the ring system, the at least two heteroatoms that are present can be identical or different. Suitable heteroaryl groups are known to the skilled person.
  • heteroaryl residues may be mentioned, as non-limiting examples: pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, and pyridazinyl.
  • alkylheteroaryl refers to heteroaryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-heteroaryl-) and which are linked via the alkyl group to the -CH2- group and via the heteroaryl group to the carbonyl group.
  • heteroarylalkyl refers to heteroaryl groups linked via the alkyl group to the carbonyl group and via the heteroaryl group to the -CH2- group (-heteroaryl-alkyl-).
  • cycloalkyl (-cycloalkyl-) means, in the context of the invention, optionally substituted, 4-10-membered cyclic alkyl residues, wherein they can be monocyclic or polycyclic groups.
  • Optionally substituted cyclohexyl may be mentioned as a preferred example of a cycloalkyl residue.
  • heterocycloalkyl refers to optionally substituted, 4-10-membered cyclic alkyl residues, which have at least one heteroatom, such as O, N or S in the ring, wherein they can be monocyclic or polycyclic groups.
  • substituted refers to residues, in which at least one H has been replaced with a suitable substituent.
  • Q 2 is an optionally substituted aryl group or optionally substituted cycloalkyl group, in particular optionally substituted phenyl or optionally substituted cyclohexyl.
  • Q 2 is preferably wherein R 11 , R 12 , R 13 and R 14 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
  • the present invention also preferably also relates to compounds having the structure
  • R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 are, independently of each other, selected from the group consisting of H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl, and wherein R 11 , R 12 , R 13 and R 14 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl. More preferably, the compound of the invention has the structure,
  • the chelator oxo-DFO* has the structure:
  • a chelator residue and typically also the term “chelator residue derived from a chelator selected from the group” is denoted to mean that the above mentioned chelators, thus typically the chelators defined in the “group”, have been linked, via a suitable group to the group -(L)n-NH-, i.e. to the residue thereby forming a covalent bond between the chelator and the compound (I).
  • the linking moiety (L)n is chosen, depending on the nature of the chelator residue A and the functional groups capable of being linked to the rest of the compound being present in A.
  • alkyl refers to a saturated acyclic hydrocarbon moiety comprising 1 to 10 carbon atoms, preferably 1-6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms, preferably 1 carbon atom.
  • aryl refers to optionally substituted 6- membered aromatic ring, i.e. optionally substituted phenyl group.
  • alkylaryl refers to aryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-aryl-) and which are linked via the alkyl group to A and via the aryl group to the rest of linker L.
  • arylalkyl refers to aryl groups linked via an alkyl group to the rest of linker L and via the aryl group to A (-aryl-alkyl-).
  • heteroaryl (-heteroaryl-), as used in this context of the invention, means optionally substituted, 5- and 6-membered aromatic rings, containing one or more, for example 1 to 4, such as 1, 2, 3, or 4, heteroatoms in the ring system. If more than one heteroatom is present in the ring system, the at least two heteroatoms that are present can be identical or different. Suitable heteroaryl groups are known to the skilled person.
  • heteroaryl residues may be mentioned, as non-limiting examples: pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, and pyridazinyl.
  • alkylheteroaryl refers to heteroaryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-heteroaryl-) and which are linked via the alkyl group to A and via the heteroaryl group to the rest of linker L.
  • heteroarylalkyl refers to heteroaryl groups linked via the alkyl group to the rest of linker L and via the heteroaryl group to A group (- heteroaryl-alkyl-).
  • substituted refers to residues, in which at least one H has been replaced with a suitable substituent.
  • n is preferably 0 and the Carboxy group is directly linked to the Amino group adjacent to group CH2-Q 2 -
  • A is a chelator residue selected from the group consisting of
  • A is a chelator residue selected from the group consisting of
  • A is a chelator residue having a structure selected from the group consisting of n is preferably 0.
  • A is
  • n is preferably 0. More preferably, the compound has the structure Pl 8
  • A is a chelator residue having a structure
  • the present invention also relates to a complex comprising
  • Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as /?-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as /?-toluenesulfonic acid, methanesulf
  • salts examples include the sulfate, pyrosulfate, bi sulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1,4- dioate, hexyne- 1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,
  • Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
  • Salts of amine groups may also comprise quaternary ammonium salts in which the amino nitrogen carries a suitable organic group such as an alkyl, alkenyl, alkynyl, or aralkyl moiety.
  • Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
  • Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
  • the potassium and sodium salt forms are particularly preferred. It should be recognized that the particular counter ion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counter ion does not contribute undesired qualities to the salt as a whole.
  • pharmaceutically acceptable solvate encompasses also suitable solvates of the compounds of the invention, wherein the compound combines with a solvent such as water, methanol, ethanol, DMSO, acetonitrile or a mixture thereof to form a suitable solvate such as the corresponding hydrate, methanolate, ethanolate, DMSO solvate or acetonitrilate.
  • a solvent such as water, methanol, ethanol, DMSO, acetonitrile or a mixture thereof.
  • the complexes of invention may contain one or more radionuclides, preferably one radionuclide.
  • These radionuclides are preferably suitable for use as radio-imaging agents or as therapeutics for the treatment of proliferating cells, for example, PSMA expressing cancer cells, in particular PSMA-expressing prostate cancer cells. According to the present invention they are called "radionuclide complexes" or "radiopharmaceuticals”.
  • Preferred imaging methods are positron emission tomography (PET) or single photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • the at least one radionuclide is selected from the group consisting 18 F, 89 Zr, 43 Sc, 44 Sc, 47 Sc, 45 Ti, 55 Co, m In, 86 Y, 90 Y, 66 Ga, 67 Ga, 68 Ga, 177 Lu, 99m Tc, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 66 Cu, 67 Cu, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 161 Tb, 166 Ho, 153 Gd, 155 Gd, 157 Gd, 211 At, 213 Bi, 225 Ac, 230 U, 223 Ra, 224 Ra, 165 Er, 52 Fe, 59 Fe, 186 Re, 188 Re, 227 Th, 133 La, 72 As and radionuclides of Pb, such as 203 Pb and 212 Pb.
  • Pb such as 203 Pb and 212 Pb.
  • the at least one radionuclide is selected from the group consisting 18 F, 68 Ga, 133 La, 177 LU, 188 Re, and 225 Ac. More preferably, the radionuclide is 18 F, 68 Ga, 177 Lu, 188 Re, and 225 Ac.
  • the radionuclide has a half-life of at least 30 min, more preferably of at least 1 h, more preferably at least 12 h, even more preferably at least Id, most preferably at least 5 d; also preferably, the radionuclide has a half-life of at most 1 year, more preferably at most 6 months, still more preferably at most 1 month, even more preferably at most 14 d.
  • the radionuclide has a half-life of from 30 min to 1 year, more preferably of 12 h to 6 months, even more preferably of from 1 d to 1 month, most preferably of from 5 d to 14 d.
  • the radionuclide according to the invention may be an a-emitter, a P -emitter, a P + -emitter, a y-emitter, and/or mixed-emitter from all options mentioned before.
  • the radionuclide is an a- and/or P-emitter, i.e. the radionuclide preferably emits a- particles (a-emitter) and/or P-radiation (P-emitter).
  • the a-particle has an energy of from 1 to 10 MeV, more preferably of from 2 to 8 MeV, most preferably of from 4 to 7 MeV.
  • the radionuclide is a P-emitter
  • the P-radiation has an energy of from 0.1 to 10 MeV, more preferably of from 0.25 to 5 MeV, most preferably of from 0.4 to 2 MeV.
  • Preferred radionuclides emitting P-radiation are selected from the group consisting of 90 Y, 177 Lu and 188 Re.
  • Preferred radionuclides emitting a -radiation are e.g. selected from the group consisting of 213 Bi, 223 Ra, 225 Ac and 227 Th.
  • the radionuclide is a positron emitter.
  • the radionuclide is preferably selected from the group consisting of 18 F, 44 Sc, 55 Co, 64 Cu, 66 Ga, 68 Ga, 72 As and 89 Zr.
  • the use is preferably PET diagnosis.
  • radionuclide is a gamma emitter.
  • the radionuclide is preferably U 1 ln or 177 Lu.
  • the use preferably is SPECT diagnosis.
  • the radionuclide emits Auger electrons, and preferably decays by electron capture.
  • the radionuclide is preferably 161 Tb.
  • the use is preferably therapy.
  • the present invention also relates to a method for preparing a compound of formula (1) or a pharmaceutically acceptable salt or solvate thereof with compound (I) being as describe above and below, the method comprising:
  • PG1 is a suitable protecting group, preferably being orthogonal to Fmoc and 2-CT linker of the resin, more preferably Alloc,
  • step (aa) Fmoc-Lys(NPG' )-OH is attached to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), the Fmoc protecting group is removed, wherein PG1 is a suitable protecting group, preferably being orthogonal to Fmoc and 2-CT linker of the resin.
  • a suitable resin preferably a 2-chloro-trityl resin (2-CT)
  • 2-CT 2-chloro-trityl resin
  • step (aa) comprises
  • Step (aal) is preferably carried out in the presence of a suitable base.
  • bases are known to the skilled person and include, in particular, organic bases, more preferably amino group comprising bases.
  • the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N- methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4- dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof.
  • step (aa) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
  • step (aal) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofuran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), di chloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
  • NMP N-methyl pyrrolidone
  • DMSO dimethyl sulfoxide
  • DMA dimethyl formamide
  • THF tetrahydrofuran
  • THF tetrahydrofuran
  • MTBE di chloromethane
  • DCM chloroform
  • tetrachloromethane chloroform
  • step (aa2) is carried out in the presence of piperidine in DMF as solvent.
  • step (aa) in particular comprises
  • the protecting group PG 1 is preferably selected from the group consisting of acetyl, trifluoroacetyl, DDE (l-(4,4-Dimethyl-2,6-dioxocyclohex-l-ylidene)-3-eth-yl), ivDDE ( 1- (4,4-Dimethyl-2,6-dioxocyclohex-l-ylidene)-3-methylbutyl) , CbZ (N-carboxybenzyl), Benzyl, Tosyl, Phtalimide, more preferably Alloc.
  • step (bb) H-G1U(OPG 2 )-OPG 2 is reacted with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming the activated Glu compound of formula (II) wherein PG 2 is a suitable protecting group, preferably being orthogonal to Fmoc and Alloc, more preferably tBu
  • the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N- methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N- methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4-dimethylaminopyridine, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (bb) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
  • DIPEA diisopropylamine
  • DIPEA A,A-ethyldiisopropylamine
  • TEA triethylamine
  • step (bb) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofiiran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), di chloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
  • NMP N-methyl pyrrolidone
  • DMSO dimethyl sulfoxide
  • DMA dimethyl formamide
  • THF tetrahydrofiiran
  • THF tetrahydrofiiran
  • 1,4-di oxane diethyl ether
  • MTBE tert. -
  • step (cc) the compound of formula (II), preferably the compound of formula (Ila), is reacted the with the Lys modified resin according to step (aa) and PG 1 is removed, thereby forming the modified resin
  • step (cc) preferably comprises
  • PG 1 is an Alloc protecting group and the group is removed by reaction with TPP palladium in the presence of morpholine.
  • step (ccl) is carried out in the presence of a suitabl base, such as piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N- methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4- dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (ccl) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
  • DIPEA diisopropylamine
  • DIPEA A,A-ethyldiisopropylamine
  • step (dd) the modified resin according to (cc) is reacted with Fmoc-(XAA 1 )-OH, and Fmoc group is removed thereby forming the modified resin
  • step (dd) preferably comprises
  • step (ddl) is carried out in the presence of a suitable activation reagent.
  • suitable activation reagents include, but are not limited to, HATU (O-(7-azabenzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate); HOAt, HBTU (O-benzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate); TBTU (2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate); TFFH (N,N',N",N"-tetramethyluronium-2-fluoro- hexafluorophosphate); BOP (benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphat
  • step (dd2) is carried out in the presence of piperidine in DMF as solvent.
  • step (ee) the modified resin according to (dd) is reacted with Fmoc- and the Fmoc group is removed.
  • step (ee) preferably comprises (eel) reacting the modified resin according to (dd) with Fmoc-
  • reaction in (ee) is preferably carried out similar to the reaction in (ddl), thus in the presence of a suitable activation reagent, Reference is made to the activation reagents mention in the context of (ddl) which are also preferred in (eel).
  • step (ff) the free amino group is linked with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L.
  • the group A-(L) n -NH- is formed.
  • step (ff) A-(L) n is preferably provided in the form of an NHS ester and is subsequently attached to free amino group.
  • A-(L) n - moiety may be linked via an amine group.
  • A-(L) n may be provided in form of an activated isothiocyanate with which the free amino group may be reacted.
  • n is preferably 1 and L is provided in the form -(Q 3 ) q -NCS, with Q 3 and q being as described above and below.
  • step (gg) the compound is cleaved from the resin and all remaining protecting groups are removed. Suitable cleaving conditions are known to the skilled person. Preferably, step (gg) is carried out in the presence of trifluoroacidic acid.
  • the present invention preferably relates to a method for preparing a resin bound PSMA binding moiety having the structure preferably more preferably more preferably as intermediate product for the solid phase synthesis of PSMA ligands, preferably for the synthesis of a PSMA ligand of as describe above and below, the method comprising the steps,
  • step Fmoc-Glu(OPG 2 )-OH is attached to a suitable resin, preferably a 2-Chloro-trityl resin (2-CT), and the Fmoc protecting group is removed, wherein PG 2 is a protecting group being orthogonal to Fmoc, preferably tBu,
  • step (a) comprises
  • Step (al) is preferably carried out in the presence of a suitable base.
  • bases are known to the skilled person and include, in particular, organic bases, more preferably amino group comprising bases.
  • the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), 7V,7V-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N- methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4- dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof.
  • step (aa) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA)
  • step (al) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofuran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), di chloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
  • step (a2) is carried out in the presence of piperidine in DMF as solvent.
  • the protecting group PG 2 is preferably selected from the group consisting of methylester, t- butylester, benzylester, S-t-butylester, more preferably tBu.
  • the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), 7V,7V-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N- methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N- methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4-dimethylaminopyridine, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (bb) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
  • DIPEA diisopropylamine
  • DIPEA 7V,7V-ethyldiisopropylamine
  • TEA triethylamine
  • step (b) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofuran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), dichloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
  • NMP N-methyl pyrrolidone
  • DMSO dimethyl sulfoxide
  • DMA dimethyl formamide
  • THF tetrahydrofuran
  • THF tetrahydrofuran
  • MTBE tert. -butyl methyl ether
  • DCM dichloromethane
  • chloroform
  • step (c) the compound of formula (II) is reacted with the Glu modified resin according to step (a) and the Fmoc group is removed, thereby forming the modified resin having the structure preferably more preferably more preferably
  • step (c) preferably comprises
  • (cl) is carried out in the presence of a base, wherein the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N-methylimidazole, 1,4- diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4-dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (bb) is carried out in the presence of N,N- ethyldiisopropylamine (DIPEA).
  • DIPEA diisopropylamine
  • DIPEA A,A-ethyldiisopropylamine
  • the resin bound PSMA binding moiety obtained by the method comprising steps (a) to (c) having the structure preferably more preferably more preferably is preferably used as intermediate product for the preparation of the PSMA ligands according to the invention described above and below.
  • the method for the preparation of the intermediate product is particularly advantageous since no hazardous reagents, such as triphosgene, is used. Further, there is no need to use an Alloc protecting group for the deprotection of which hazardous TPP palladium in the presence of morpholine needs to be used.
  • the present invention thus also relates to a method for preparing a compound of formula (1) in which the intermediate compound is prepared according to steps (a) to (c) described above and which is then transformed in further reaction steps to a PSMA ligand according to the invention.
  • the present invention also relates to method for preparing a compound of formula (1)
  • step (e) the modified resin according to (d) is reacted with Fmoc and the Fmoc group is removed.
  • step (e) preferably comprises
  • reaction in (e) is preferably carried out similar to in the presence of a suitable activation reagent, Reference is made to the activation reagents mention in the context of (ddl) which are also preferred in (el).
  • step (el) is carried out in the presence of HBTU or Oxyma Pure and DIC.
  • step (e2) is carried out in the presence of piperidine in DMF as solvent.
  • step (ff) A-(L) n is preferably provided in the form of an NHS ester and is subsequently attached to free amino group.
  • A-(L) n - moiety may be linked via an amine group.
  • A-(L) n may be provided in form of an activated isothiocyanate with which the free amino group may be reacted.
  • n is preferably 1 and L is provided in the form -(Q 3 ) q -NCS, with Q 3 and q being as described above and below.
  • step (g) the compound is cleaved from the resin and all remaining protecting groups are removed. Suitable cleaving conditions are known to the skilled person. Preferably, step (g) is carried out in the presence of trifluoroacidic acid.
  • the present invention also relates to a compound of formula (1), obtained or obtainable by a method according described herein.
  • Pharmaceutical compositions and therapeutic uses are also relates to a compound of formula (1), obtained or obtainable by a method according described herein.
  • the present invention relates to a pharmaceutical composition comprising a compound or a complex as specified herein. Further, the present invention relates to a compound, a complex, or a pharmaceutical composition as described herein for use in medicine, in particular for use in treating and/or preventing a PSMA expressing cancer, in particular prostate cancer and/or metastases thereof.
  • the present invention also relates to a method for treating a PSMA expressing cancer in a patient, the method comprising (A) contacting said patient with a compound, a complex, or a pharmaceutical composition as described herein to said sample; and (B) thereby treating said cancer in said patient.
  • the present invention also relates to a use of a compound, a complex, or a pharmaceutical composition as described herein for the manufacture of a medicament for treating a PSMA expressing cancer.
  • composition and “pharmaceutical composition” are used essentially interchangeably herein and are, in principle, known to the skilled person. As referred to herein, the terms relate to any composition of matter comprising the specified active agent(s) as pharmaceutically active compound(s) and one or more excipient.
  • the pharmaceutically active compound(s) can be present in liquid or dry, e.g. lyophilized, form. It will be appreciated that the form and character of the pharmaceutical acceptable excipient, e.g. carrier or diluent, is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables.
  • the excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof.
  • the excipient employed may include a solid, a gel, or a liquid.
  • Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • Exemplary of liquid carriers are phosphate buffered saline solution, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution, syrup, oil, water, emulsions, various types of wetting agents, and the like.
  • the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
  • suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.
  • the excipient(s) is/are selected so as not to affect the biological activity of the combination.
  • the excipient may, however, also be selected to improve uptake of the active agent into a host cell, in particular a target cell.
  • the medicament is, typically, administered systemically, preferably orally or parenterally, e.g. by intravenous administration, or is administered topically, preferably intra- tumorally, topically on a body surface, or by inhalation; in case of cancer treatment, topical administration may be intratumoral or peritumoral, and/or topical at a site of tumor excision. Administration may, however, also be into a blood vessel, typically an artery, afferent to an intended site of effect, such as a tumor. However, depending on the nature of the formulation and the desired therapeutic application, the medicament may be administered by other routes as well.
  • the medicament is preferably administered in conventional dosage forms prepared by combining the drug with standard pharmaceutical carriers according to conventional procedures.
  • a therapeutically effective dose refers to an amount of the active substance to be used in a medicament which prevents, ameliorates or cures the symptoms accompanying a disease or condition referred to in this specification.
  • Therapeutic efficacy and toxicity of a drug can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • ED50 the dose therapeutically effective in 50% of the population
  • LD50 the dose lethal to 50% of the population.
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • the dosage regimen will be determined by the attending physician and by clinical factors.
  • dosages for any one patient depends upon many factors, including the patient's size, age, the particular formulation of the medicament to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • the medicament referred to herein is, preferably, administered at least once, e.g. as a bolus. However, the medicament may be administered more than one time and, preferably, at least twice, e.g. permanently or periodically after defined time windows. Progress can be monitored by periodic assessment. Dosage recommendations may be indicated in the prescriber or user instructions in order to anticipate dose adjustments depending on the considered recipient. Typical doses for compounds similar to those of described herein, e.g. PSMA-617, are known in the art, e.g. from WO 2020/165420. In view of the pharmacokinetic properties of the compounds described herein, essentially the same dosages are applicable for these as well.
  • the medicament according to the present invention may comprise further active agents in addition to the aforementioned active agent(s).
  • the pharmaceutically active compound according to the invention is to be applied together with at least one further drug and, thus, may be formulated together with this at least one further drug as a medicament.
  • said at least one further active agent is a chemotherapeutic agent or an immunotherapeutic agent, such as an immune checkpoint modulator.
  • the formulation of a pharmaceutical composition preferably takes place under GMP standardized conditions or the like in order to ensure quality, pharmaceutical safety, and effectiveness of the medicament.
  • cancer relates to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells (“cancer cells”). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue and possibly spread of cancer cells to other locations in the body (metastasis).
  • cancer is a relapse.
  • the cancer is a solid cancer, a metastasis, or a relapse thereof.
  • the cancer is an uncontrolled proliferation of cells comprising cells expressing PSMA.
  • the cancer is a solid cancer comprising cells expressing PSMA, e.g. cells forming blood vessels, preferably in neovasculature.
  • the cancer may in particular be a PSMA expressing cancer such as prostate cancer, thyroid cancer, salivary gland cancer, lung cancer, liver cancer, kidney cancer, breast cancer, or brain cancer, or a metastasis of any of the aforesaid. More preferably, the cancer is prostate cancer and/or a metastasis thereof
  • the term "patient”, as used herein, relates to a vertebrate, preferably a mammalian animal, more preferably a human, monkey, cow, horse, cat or dog.
  • the mammal is a primate, more preferably a monkey, most preferably a human.
  • the patient is known or suspected to suffer from a cancer as specified herein.
  • treating refers to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of health with respect to the diseases or disorders referred to herein. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, as specified herein above.
  • prevention refers to retaining health with respect to the diseases or disorders referred to herein for a certain period of time in a subject. It will be understood that the said period of time may be dependent on the amount of the drug compound which has been administered and individual factors of the subject discussed elsewhere in this specification. It is to be understood that prevention may not be effective in all subjects treated with the compound according to the present invention. However, the term requires that, preferably, a statistically significant portion of subjects of a cohort or population are effectively prevented from suffering from a disease or disorder referred to herein or its accompanying symptoms. Preferably, a cohort or population of subjects is envisaged in this context which normally, i.e.
  • treatment and/or prevention comprises administration of at least one compound as specified herein and/or at least one complex as specified elsewhere herein.
  • the invention provides a method for treating a patient suffering from cancer by administering to said patient a therapeutically effective amount of a compound, complex, or medicament, all as described above or below, to treat a patient suffering from a cancer.
  • the present invention also relates to a compound as described herein, a complex as described herein, or a pharmaceutical composition as described herein, for use in diagnostics, in particular for use in the diagnosis of cancer, preferably of PSMA expressing cancer, in particular of prostate cancer and/or metastases thereof.
  • diagnostics in particular for use in the diagnosis of cancer, preferably of PSMA expressing cancer, in particular of prostate cancer and/or metastases thereof.
  • Said diagnostic methods preferably are practised on the human or animal body.
  • the present invention relates to a compound as described herein, a complex as described herein, or a pharmaceutical composition as described herein for use in diagnosing a PSMA expressing cancer in a patient, said diagnosing comprising (i) determining binding of said compound or complex to a tissue of said subject; (ii) comparing the binding determined in step (i) to a reference; (iii) identifying a difference in step (ii); and (iv) diagnosing said cancer based on the result of step (iii).
  • the present invention also relates to a method for diagnosing a PSMA expressing cancer in a sample of a patient, the method comprising (I) determining binding of a compound, a complex, or a pharmaceutical composition as described herein to said sample; and (II) diagnosing said cancer based on the result of step (I); said method may comprise further step (la) comparing the binding determined in step (I) to a reference; in such case, step (II) preferably comprises diagnosing said cancer based on the result of step (la), more preferably is step (Ila) diagnosing said cancer based on the result of step (la).
  • the present invention also relates to a use of a compound, a complex, or a pharmaceutical composition as described herein for the manufacture of a diagnostic for diagnosing a PSMA expressing cancer.
  • diagnosis refers to assessing whether a subject suffers from a disease or disorder, preferably cell proliferative disease or disorder, or not. As will be understood by those skilled in the art, such an assessment, although preferred to be, may usually not be correct for 100% of the investigated subjects. The term, however, requires that a, preferably statistically significant, portion of subjects can be correctly assessed and, thus, diagnosed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.
  • the p-values are, preferably, 0.2, 0.1, or 0.05.
  • diagnosing may comprise further diagnostic assessments, such as visual and/or manual inspection, determination of tumor biomarker concentrations in a sample of the subject, X-ray examination, and the like. The term includes individual diagnosis of as well as continuous monitoring of a patient. Monitoring, i.e.
  • diagnosing the presence or absence of cell proliferative disease or the symptoms accompanying it at various time points includes monitoring of patients known to suffer from cancer as well as monitoring of subjects known to be at risk of developing cell proliferative disease. Furthermore, monitoring can also be used to determine whether a patient is treated successfully or whether at least symptoms of cell proliferative disease can be ameliorated over time by a certain therapy. Moreover, the term also includes classifying a subject according to a usual classification scheme, e.g. the T1 to T4 staging, which is known to the skilled person.
  • the compound, as described above or below, or the complex, as described above or below, or the pharmaceutical composition, as described above or below, are used for in vivo imaging and radiotherapy.
  • Suitable pharmaceutical compositions may contain a radio imaging agent, or a radiotherapeutic agent that has a radionuclide either as an element, i.e. radioactive iodine, or a radioactive metal chelate complex of the compound of formula (la) and/or (lb) in an amount sufficient for imaging, together with a pharmaceutically acceptable radiological vehicle.
  • the radiological vehicle should be suitable for injection or aspiration, such as human serum albumin; aqueous buffer solutions, phosphate, citrate, bicarbonate, etc; sterile water physiological saline; and balanced ionic solutions containing chloride and or dicarbonate salts or normal blood plasma cautions such as calcium potassium, sodium and magnesium.
  • concentration of the imaging agent or the therapeutic agent in the radiological vehicle should be sufficient to provide satisfactory imaging. Appropriate dosages have been described e.g. in WO 2020/165420.
  • sample refers to a sample of a bodily fluid, preferably a sample of blood, plasma, serum, saliva, sputum, urine, or a sample comprising separated cells or to a sample from a tissue or an organ.
  • the sample is a cancer sample, in particular a tumor or metastasis sample, which may be obtained e.g. by biopsy.
  • a cancer sample in particular a tumor or metastasis sample, which may be obtained e.g. by biopsy.
  • Separated cells may be obtained from the body fluids, such as lymph, blood, plasma, serum, liquor and other, or from the tissues or organs by separating techniques such as centrifugation or cell sorting.
  • the sample is a sample known or suspected to comprise cancer cells.
  • the sample can be obtained from the patient by any method deemed appropriate by the skilled person, preferably by routine techniques which are well known to the person skilled in the art, e.g., venous or arterial puncture or open biopsy including aspiration of tissue or cellular material from a subject. For those areas which cannot be easily reached via an open biopsy, a surgery and, preferably, minimal invasive surgery can be performed.
  • Q 2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
  • A is a chelator residue derived from a chelator selected from the group consisting of
  • Q 2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
  • A is a chelator residue derived from a chelator selected from the group consisting of
  • step (cc) reacting the compound of formula (II) with the Lys modified resin according to step (aa) and removing PG 1 , thereby forming the modified resin
  • step (gg) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
  • step (aa) comprises
  • step (aa2) removing the Fmoc group in the presence of piperidine in DMF. 15.
  • step (bb) is carried out in the presence of a base, preferably DIPEA.
  • step (ee) comprises
  • (ee2) removing the Fmoc group. wherein (eel) is carried out in the presence of a suitable activation reagent, preferably HBTU or Oxyma Pure and DIC.
  • a suitable activation reagent preferably HBTU or Oxyma Pure and DIC.
  • step (gg) is carried out in the presence of trifluoroacidic acid.
  • Q 2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
  • A is a chelator residue derived from a chelator selected from the group consisting of
  • DOTAM N,N'-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N'-diacetic acid
  • H4Octapa 1,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid
  • PEPA cyclam-based ligands with N-methyl, N-(4-aminobenzyl), N-phosphinate- and phosphonate-based substituents, wherein oxo-DFO* has the structure: the method comprising:
  • step (g) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
  • step (a) comprises
  • step (a2) removing the Fmoc group in the presence of piperidine in DMF.
  • step (b) is carried out in the presence of a base, preferably DIPEA.
  • a base preferably DIPEA.
  • step (c) comprises
  • composition comprising a compound of any one of embodiment 1 to0 or a complex of embodiment 11 or 12. 31.
  • step (ii) comparing the binding determined in step (i) to a reference
  • step (iii) identifying a difference in step (ii);
  • step (iv) diagnosing said cancer based on the result of step (iii).
  • a method for diagnosing a PSMA expressing cancer in a sample of a patient comprising
  • step (II) diagnosing said cancer based on the result of step (I).
  • step (la) comparing the binding determined in step (I) to a reference; wherein step (II) comprises diagnosing said cancer based on the result of step (la).
  • a method for treating a PSMA expressing cancer in a patient comprising
  • Fig. 1 Synthetic scheme for the conjugation of Fmoc-TXA for Pl 7.
  • Fig. 2 Synthetic scheme for the conjugation of the DOTA chelator to obtain P17/P18.
  • Fig. 3 Synthesis Scheme for the Synthesis ofP17 via method 1.
  • Fig. 4 Synthesis Scheme for the Synthesis of P18 via method 1.
  • Fig. 5 Synthesis of urea intermediate via method 2.
  • Fig. 6 Synthesis Scheme for the Synthesis of P17 via method 2.
  • Fig. 7 Synthesis Scheme for the Synthesis of P18 via method 2.
  • Fig. 8 Chemical structures of (a) P17 and (b) P17-Macropa.
  • Fig. 9 Chemical structures of (a) Pl 8, (b) P18-Macropa, and (c) P18-HEHA.
  • Fig. 10 Chemical structure of PSMA-617.
  • Fig. 11 Structural characterization of PSMA complexes.
  • A The Fo-Fc omit map (gray) is contoured at 3.0 c and inhibitors are shown in stick representation. The active-site zinc ions are shown as black spheres. Notice the absent electron density for the DOTA chelator of 617 implying its positional flexibility due to missing interactions with PSMA.
  • B Superposition of inhibitors in PSMA/inhibitor complexes. PSMA molecules in individual complexes were superpositioned on corresponding Ca atoms. While positioning of P17/P18 is virtually identical, there are marked differences in positioning of linker and chelator moieties of P17 (gray) and 617 (black).
  • Fig. 12 Graphs representing the uptake of radiolabeled PSMA compounds in PC3-PIP and PC3-flu tumors and various other tissues and organs at 1 h and 2 h after injection.
  • A [ 177 Lu]Lu- P17;
  • B [ 177 LU]LU-P18;
  • C [ 177 LU]LU-PSMA-617.
  • Scheme 1A Synthesis of the peptidomimetic glutamate-urea-lysine binding motif via analogical strategy as the one for PSMA-617
  • 2-Chloro-trityl resin (2-CT resin, 0.3 mmol, substitution capacity 1.22 mmol/g, 100-200 MESH) was first agitated in dry dichloromethane (DCM) for 45 min and then washed with dry DCM followed by reaction with 1.2 equiv Alloc- (As-allyloxycarbonyl) and Fmoc (Na- fhiorenylmethoxycarbonyl)-protected L-lysine [Fmoc-L-Lys(Alloc)-OH, 1], and 4.8 equiv A,A-ethyldiisopropylamine (DIPEA) in 3 mL of dry DCM. The coupling of the first protected amino acid on the resin proceeded over the course of 16 h with gentle agitation.
  • DIPEA 2-Chloro-trityl resin
  • the lysine-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, methanol (MeOH), and DIPEA in a ratio of 17:2: 1, respectively.
  • Selective removal of the Fmoc-protecting group was realized by washing with a mixture of A,A-dimethylformamide (DMF) and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to get product 3.
  • DMF A,A-dimethylformamide
  • the product 4 was filtered off and washed with DCM.
  • the cleavage of the Alloc-protecting group was realized by reaction with 0.3 equiv tetrakis(triphenyl)palladium (TPP palladium) in the presence of 30 equiv morpholine in dry DCM for 2 h.
  • the reaction was performed in the dark using aluminum foil.
  • the resin was washed with 1% DIPEA in DMF and subsequently with a solution of sodium diethyldithiocarbamate trihydrate (15 mg/mL) in DMF (10 times for 5 min).
  • the resin- immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
  • the lysine-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, MeOH, and DIPEA in a ratio of 17:2: 1, respectively.
  • Selective removal of the Fmoc-protecting group was realized by washing three times with 30 mL of a mixture of DMF and piperidine in a ratio of 1 : 1 in order to get product 3.
  • the product 4 was filtered off and washed with DCM.
  • the cleavage of the Alloc-protecting group was realized by reaction with 0.3 equiv TPP palladium in the presence of 30 equiv morpholine in dry DCM for 2 h. The reaction was performed in the dark using aluminum foil.
  • the resin was washed with 1% DIPEA in DMF and subsequently with a solution of sodium diethyldithiocarbamate trihydrate (15 mg/mL) in DMF.
  • the resin-immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
  • Scheme 1C Synthesis of the peptidomimetic glutamate-urea-lysine binding motif via adapted new strategy
  • the lysine-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, MeOH, and DIPEA in a ratio of 17:2:1, respectively.
  • Selective removal of the Fmoc-protecting group was realized by washing three times with 30 mL of a mixture of DMF and piperidine in a ratio of 1 : 1 in order to get product 3.
  • the product 4 was filtered off and washed with DCM.
  • the cleavage of the Alloc-protecting group was realized by reaction with 0.3 equiv TPP palladium in the presence of 30 equiv morpholine in dry DCM for 2 h. The reaction was performed in the dark using aluminum foil.
  • the resin was washed with 1% DIPEA in DMF and subsequently with a solution of sodium diethyldithiocarbamate trihydrate (15 mg/mL) in DMF.
  • the resin-immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
  • the glutamate-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, MeOH, and DIPEA in a ratio of 17:2: 1, respectively.
  • Selective removal of the Fmoc-protecting group was realized by washing three times with 30 mL of a mixture of DMF and piperidine in a ratio of 1 : 1 in order to get product 3.
  • the product 4 was filtered off and washed with DCM and DMF. Selective removal of the Fmoc- protecting group was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min.
  • the resin-immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
  • Scheme 2A Coupling of linker binding blocks via analogical strategy as the one for PSMA- 617
  • Scheme 2AA Coupling of linker binding blocks via analogical strategy as the one for PSMA- 617
  • Scheme 2AAA Coupling of linker binding blocks via analogical strategy as the one for PSMA-
  • Scheme 3C Conjugation of DOTA-//7.s(/Bu)ester via analogical strategy as the one for PSMA- 617 Relative to the resin (0.1 mmol), 4 equiv DOTA-//7.s(/Bu)ester (2-(4,7,10-tris(2-(t-butoxy)-2- oxoethyl)-l,4,7,10-tetraazacyclododecan-l-yl)acetic acid, 0.400 mmol) were activated with 3.95 equiv HBTU (0.395 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF.
  • amine precursor 50 mg was dissolved in 2 mL DMF. After the addition of 25 mg HEHA-NCS ((4-isothiocyanatobenzyl)-l,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid) and 30 pL DIPEA, the reaction was stirred for 16 h at room temperature. After the evaporation of the solvent at 60 °C, the reaction mixture was diluted with water and ACN to a volume of 4 mL, purified by HILIC -HPLC and analyzed by ESI-MS.
  • HEHA-NCS ((4-isothiocyanatobenzyl)-l,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid)
  • DIPEA 30 pL DIPEA
  • P17/P18 were analyzed by RP-HPLC, electrospray ionization mass spectrometry (ESI-MS) as well as ID 'H and 13 C nuclear magnetic resonance (NMR), and 2D NMR techniques, viz. correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC).
  • ESI-MS electrospray ionization mass spectrometry
  • NMR nuclear magnetic resonance
  • 2D NMR techniques viz. correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC).
  • COSY correlation spectroscopy
  • HSQC heteronuclear single quantum correlation
  • HMBC heteronuclear multiple bond correlation
  • No-carrier-added lutetium-177 [ 177 Lu]LuCh; ITM Medical Isotopes GmbH, Germany
  • sodium acetate 0.4 M, pH 4.5
  • the respective PSMA ligand (1 mM stock solution in DMSO) was added to obtain a molar activity of 1-10 MBq/nmol (dependent on the experiment) and the reaction mixture was incubated for 30 min at 95 °C. Quality control was performed by TLC and HPLC analysis as previously reported. The radiolabeling efficiency was >95% for all tested PSMA ligands.
  • PSMA prostate-specific membrane antigen
  • Diffraction data were collected from a single crystal using synchrotron radiation at the MX14.1 and MX14.2 beamlines (0.91841 A; BESSYII, Berlin, Germany) equipped with a Pilatus 6M detector (Dectris, Switzerland) at 100 K. Datasets were indexed, integrated and scaled using the XDSAPP interface (Sparta et al., 2016).
  • the wide diameter and partial flexibility of the PSMA entrance funnel allows for the 2-naphtylic function to fold inward and to push the aminohexanoyl group to the opposite wall of the funnel, effectively thus filling its lower portion.
  • the corresponding less lipophilic 3-styryl moiety in P17/P18 is oriented towards the exterior of the protein (Fig. 1 IB) where its phenyl ring is sandwiched between the Lys207 side chain (s-NFL. . .ring center, 3.9 A) and the DOTA function (4.0 A).
  • variable positioning of distal parts of inhibitors is facilitated by the structural plasticity of the “entrance lid”, the flexible amino acid stretch comprising residues Thr538 - Gly548 of PSMA.
  • the lid adopts the “semi-closed” conformation, and the linker moieties follow the contours of the lid with the 2-naphtyl group engaged in CH/K interactions with Ca carbons of Ser547 (4.5 A) and Gly548 (3.4 A), while the adjacent cyclohexanoyl ring (in the chair conformation) is tightly packed against the phenyl ring of Phe546 with the distance of 5.1 A between the ring centers.
  • the lid is pushed away from the center of the entrance funnel (up to 6.2 A for corresponding Phe546-Ca carbons) accommodating thus the less compact conformation of the inhibitor linker (Fig. 1 IF).
  • the Fo-Fc electron density peaks are missing for the DOTA chelator of 617, and this observation is consistent with the absence of intermolecular interactions with PSMA and ensuing positional flexibility outside the entrance funnel of PSMA.
  • the DOTA function is well resolved in Fo-Fc maps of PSMA/P17/P18 complexes, where its position is stabilized via an array of CH/K interactions between the methylene groups of the central 12-membered tetraazacyclododecane ring and the indole group of the Trp541 side chain of the entrance lid (Fig. 11 A, G).
  • HPLC separation was performed over an 11-min linear gradient from 20 to 50 % of eluent B (0.1% (v/v) TFA in acetonitrile) at a flow rate of 0.6 mL/min.
  • the mobile phase A was 0.1% (v/v) TFA in water.
  • the non-inhibited reaction was assigned to 100%, and the reaction without an enzyme was defined as 0%.
  • the data were fitted using the GraphPad Prism software and IC50 values were calculated from the inhibition curves of three independent experiments using a nonlinear regression protocol.
  • Potency of studied inhibitors against recombinant human PSMA was determined in vitro using an HPLC-based assay with fhioresceine-y-Glu-Glu as a substrate and calculated IC50 values were 145, 344, and 128 pM for 617, P17, and P18, respectively.
  • the subnanomolar inhibitory potencies are virtually identical for the parent 617 and P17/P18 derivatives and reflect tight binding to PSMA via extensive contacts as observed in our X-ray structures of PSMA/inhibitor complexes (above).
  • affinities of metal-loaded P17/P18 inhibitors for PSMA are lower compared to metal-free counterparts and trends in binding affinities correlate with the metal coordination via carboxylic pendants (binding affinity better for natGa involving only two pendants than for natBi/natLa involving all four) as well as size of the metal (binding affinity worse for natLa with 195 ppm than for natBi with 160 ppm and natGa with 130 ppm) —> IC50 L > IC50 [natGa]-L > [natBi]-L > [natLa]-L.
  • the binding affinities of PSMA-617, P17 and Pl 8 were determined using a competitive assay with [ 177 LU]LU-PSMA-617. All three compounds were incubated at 12 different concentrations (0, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 500, 1000 and 5000 nM) with 0.75 nM [ 177 Lu]Lu-PSMA-617 on either LNCaP (PSMA+), C4-2 (PSMA+), PC3-PIP (PSMA+) or PC3-flu (PSMA-) cells. After 45 min incubation at the RT, the activity was removed and cells were washed twice with 100 pL and once with 200 pL ice-cold PBS.
  • the activity specifically accumulated in the cells was measured in a gamma counter (Cobra Autogamma B5003, Canberra, Packard; Frankfurt, Germany). The data were fitted using a non-linear regression algorithm (GraphPad Prism Software, version 8) to calculate 50% inhibitory concentrations (IC50 values). The experiment was repeated in three independent assays performed in a quadruplicate and the data are presented as the average ⁇ SD. Statistical analysis using multiple unpaired t-test (GraphPad Prism Software, version 8) was pursued and a p- value of ⁇ 0.05 was considered statistically significant.
  • the cells were incubated for 45 min at 37 °C with 30 nM [ 177 Lu]Lu-P17, [ 177 Lu]Lu-P18 or [ 177 Lu]Lu-PSMA-617 in 250 pL Opti-MEM (Gibco). Moreover, another set of cells was treated analogically while the PSMA-inhibitor 2- (phosphonomethyl)pentane- 1,5 -dioic acid (2-PMPA; 100 mM, 500 pM/well) was added in order to subtract the non-specific binding.
  • 2-PMPA phosphonomethylpentane- 1,5 -dioic acid
  • the activity was removed and cells were washed three times with 1 mL ice-cold PBS and the surface-bound radioactivity was removed by incubating the cells twice with 500 pL glycine buffer (50mM; pH 2.8) for 5 min. After washing the cells once with 1 mL ice-cold PBS, the internalized fraction was determined by subsequent cells lysis with 500 pL NaOH (0.3M; pH 14). The collected glycine and hydroxide fractions were measured in a gamma counter (Cobra Autogamma B5003, Canberra, Packard; Frankfurt, Germany) and calculated as percentage of total applied activity specifically bound on either cells surface or internalized inside the cells.
  • a gamma counter Cobra Autogamma B5003, Canberra, Packard; Frankfurt, Germany
  • Tumor-bearing mice were intravenously injected with either [ 177 Lu]Lu-PSMA-617, [ 177 Lu]Lu- P17 and [ 177 Lu]Lu-P18. The mice were sacrificed at 1 h, 2 h and 24 h post-injection (p.i.). Groups of 3 mice were used for each time-point. Selected tissues and organs were collected, weighed, and measured in a gamma counter (Cobra Autogamma B5003, Canberra, Packard; Frankfurt, Germany). The results were calculated as percentage of the injected activity per gram of tissue mass (% lA/g) and the data are presented as the average ⁇ SD.
  • Table 2 IC50 values determined via GCPILbased enzymatic assay.
  • Table 3 IC50 values determined via PSMA cell-based assay.
  • Table 4 Internalization rate. Table 5. Biodistribution data obtained in PC3-PIP and PC3-flu tumor-bearing balb/c nude mice at 1, 2 and 24 h after injection of [ 177 Lu]Lu-P17. Salivary glands 0.23 ⁇ 0.04 0.10 ⁇ 0.02 0.03 ⁇ 0.01

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Abstract

The present invention generally relates to the field of radiopharmaceuticals and their use in nuclear medicine as tracers, imaging agents and radiopharmaceuticals for the treatment of various disease states of PSMA-expressing cancers, especially prostate cancer, and metastases thereof. Further the present invention provides synthetic procedures for the synthesis of such PSMA-targeting ligands.

Description

THERANOSTIC PSMA-TARGETING LIGANDS FOR THE DIAGNOSIS AND TREATMENT OF PSMA-EXPRESSING CANCERS AND THEIR SOLID PHASE SYNTHESIS
Field of the invention
The present invention generally relates to the field of radiopharmaceuticals and their use in nuclear medicine as tracers, imaging agents and radiopharmaceuticals for the treatment of various disease states of PSMA-expressing cancers, especially prostate cancer, and metastases thereof. Further the present invention provides synthetic procedures for the synthesis of such PSMA-targeting ligands,
Prostate cancer (PCa) is the leading cancer in the US and European population in men. At least 1-2 million men in the western hemisphere suffer from prostate cancer and it is estimated that the disease will strike one in six men between the ages of 55 and 85. There are more than 300,000 new cases of prostate cancer diagnosed each year in USA. The mortality from the disease is second only to lung cancer. Currently, imaging methods with high resolution of the anatomy, such as computed tomography (CT), magnetic resonance (MR) imaging and ultrasound, predominate for clinical imaging of prostate cancer. An estimated annual $ 2 billion is currently spent worldwide on surgical, radiation, drug therapy and minimally invasive treatments. However, there is presently no effective therapy for relapsing, metastatic, androgenindependent prostate cancer.
A variety of experimental low molecular weight PCa imaging agents are currently being pursued clinically, including radiolabeled choline analogs [18F]fluorodihydrotestosterone ([18F]FDHT), anti-l-amino-3-[18F]fluorocyclobutyl-l-carboxylic acid (anti[18F]FACBC), [nC]acetate and l-(2-deoxy-2-[18F]flouro-L-arabinofuranosyl)-5-methyluracil ([18F]FMAU) (Scher, B.; et al. Eur J Nucl Med Mol Imaging 2007, 34, 45-53; Rinnab, L; et al. BJU Int 2007, 100, 786,793; Reske, S.N.; et al. J Nucl Med 2006, 47, 1249-1254; Zophel, K.; Kotzerke, J. Eur J Nucl Med Mol Imaging 2004, 31, 756-759; Vees, H.; et al. BJU Int 2007, 99, 1415-1420; Larson, S. M.; et al. J Nucl Med 2004, 45, 366-373; Schuster, D.M.; et al. J Nucl Med 2007, 48, 56-63; Tehrani, O.S.; et al. J Nucl Med 2007, 48, 1436-1441). Each operates by a different mechanism and has certain advantages, e.g., low urinary excretion for [nC]choline, and disadvantages, such as low specificity.
It is well known that tumors may express unique proteins associated with their malignant phenotype or may over-express normal constituent proteins in greater number than normal cells. The expression of distinct proteins on the surface of tumor cells offers the opportunity to diagnose and characterize disease by probing the phenotypic identity and biochemical composition and activity of the tumor. Radioactive molecules that selectively bind to specific tumor cell surface proteins provide an attractive route for imaging and treating tumors under non-invasive conditions. A promising new series of low molecular weight imaging agents targets the prostate-specific membrane antigen (PSMA) (Mease R.C. et al. Clin Cancer Res. 2008, 14, 3036-3043; Foss, C.A.; et al. Clin Cancer Res 2005, 11 , 4022-4028; Pomper, M.G.; et al. Mol Imaging 2002, 1 , 96-101; Zhou, J.; et al. Nat Rev Drug Discov 2005, 4, 015-1026; WO 2013/022797).
PSMA is a trans-membrane, 750 amino acid type II glycoprotein that has abundant and restricted expression on the surface of PCa, particularly in androgen-independent, advanced and metastatic disease (Schulke, N.; et al. Proc Natl Acad Sci U S A 2003, 100, 12590-12595). The latter is important since almost all PCa become androgen independent over the time. PSMA possesses the criteria of a promising target for therapy (Schulke, N.; et al. Proc. Natl. Acad. Sci. U S A 2003, 100, 12590-12595). The PSMA gene is located on the short arm of chromosome 11 and functions both as a folate hydrolase and neuropeptidase. It has neuropeptidase function that is equivalent to glutamate carboxypeptidase II (GCPII), which is referred to as the "brain PSMA", and may modulate glutamatergic transmission by cleaving N-acetyl aspartyl glutamate (NAAG) to N-acetyl aspartate (NAA) and glutamate (Nan, F.; et al. J Med Chem 2000, 43, 772- 774). There are up to 106 PSMA molecules per cancer cell, further suggesting it as an ideal target for imaging and therapy with radionuclide-based techniques (Tasch, J.; et al. Crit Rev Immunol 2001, 21, 249-261).
The radio-immunoconjugate of the anti-PSMA monoclonal antibody (mAb) 7E11, known as the PROSTASCINT® scan, is currently being used to diagnose prostate cancer metastasis and recurrence. However, this agent tends to produce images that are challenging to interpret (Lange, P.H. PROSTASCINT® scan for staging prostate cancer. Urology 2001 , 57, 402-406; Haseman, M.K.; et al. Cancer Biother Radiopharm 2000, 15, 131-140; Rosenthal, S.A.; et al. Tech Urol 2001 , 7, 27-37). More recently, monoclonal antibodies have been developed that bind to the extracellular domain of PSMA and have been radiolabeled and shown to accumulate in PSMA-positive prostate tumor models in animals. However, diagnosis and tumor detection using monoclonal antibodies has been limited by the low permeability of the monoclonal antibody in solid tumors.
The selective targeting of cancer cells with radiopharmaceuticals, either for imaging or therapeutic purposes is challenging. A variety of radionuclides are known to be useful for radioimaging or cancer radiotherapy, including U 1ln, 90Y, 68Ga, 177Lu, "mTc, 123I and 131I. Recently it has been shown that some compounds containing a glutamate-urea-glutamate (GUG) or a glutamate-urea-lysine (GUL) recognition element linked to a radionuclide-ligand conjugate exhibit high affinity for PSMA.
In WO 2015/055318 new imaging agents with improved tumor targeting properties and pharmacokinetics were described. These compounds comprise a motif specifically binding to cell membranes of cancerous cells, wherein said motif comprises a prostate-specific membrane antigen (PSMA), that is the above mentioned glutamate-urea-lysine motif. The preferred molecules described in WO 2015/055318 further comprise a linker which binds via an amide bond to a carboxylic acid group of DOTA as chelator. Some of these compounds have been shown to be promising agents for the specific targeting of prostate tumors. The compounds were labeled with 177Lu (for therapy purposes) or 68Ga (for diagnostic purposes) and allow for visualization and targeting of prostate cancer for radiotherapy purposes. Among the most promising compounds, MB- 17 (or PSMA-617) was disclosed. The ligand MB-24 (or PSMA-624) disclosed in WO 2015/055318 and comprising an aromatic linker moiety instead of the cyclohexyl moiety present in PSMA-617 demonstrated a high internalization rate. However, it also showed higher retention in non-targeted organs and tissues in comparison with PSMA-617. See also Benesova M, Bauder-Wiist U, Schafer M, Klika KD, Mier W, Haberkorn U, Kopka K, Eder M. Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors. J Med Chem. 2016 Mar 10;59(5): 1761- 75. DOI: 10.1021/acs.jmedchem.5b01210.
However, there is still the need for further PSMA ligands which enable various strategies for the detection, treatment and management of PSMA-expressing cancers, in particular prostate cancer.
Further, the synthesis of the binding motif described in the prior art is carried out via a solid phase approach employing hazardous triphosgene, which generally limit the solid phase synthetic approach for PSMA ligands. Furthermore, the method used so far is incompatible with a large number of synthetic building blocks to be used due to solubility problems.
Thus, there is the need for improved synthetic procedures, in particular procedures which avoid the use of hazardous reagents, such as triphosgene, and which extend and improve the synthetic options in particular when using insoluble building blocks. Thus, there is also the need for improved synthetic methods for the preparation of PSMA ligands.
Summary of the invention
The solution of said object is achieved by providing the embodiments characterized in the claims. The inventors found new compounds which are useful and advantageous radiopharmaceuticals and which can be used in nuclear medicine as tracers, imaging agents and for the treatment of various disease states of PSMA-expressing cancers, in particular prostate cancer. These compounds are described in more detail below.
Further, the inventors have found improved methods for preparing said compounds, which are carried out without the need for hazardous components such as triphosgene and which extend and improve the synthetic options. These methods are also described in more detail below.
In particular, the present invention relates to a compound of formula (1)
Figure imgf000004_0001
or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is H or -CH3, preferably H, wherein R2, R3 and R4 are independently of each other, selected from the group consisting of - CO2H, -SO2H, -SO3H, -OSO3H, -PO2H2, -PO3H2, -OPO3H2, -CONH2 and -C0NH0H,. wherein L is a suitable linker, linking A and NH, preferably L is a linking group selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q-C(=O)-, -(Q3)q-C(=S)-, -(Q3)q-NH-C(=S)-, wherein Q3 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is 0 or 1, and wherein n is 0 or 1, wherein XAA1 is an amino acid moiety having the structure (A)
Figure imgf000005_0001
with Q1 being a residue comprising an optionally substituted styryl moiety, said residue preferably having the structure -(CHR5)m-CH=CH-Ph, where m equals 0, 1, 2, or 3, each R5, independently of others, is selected from the group consisting of H, alkyl and aryl, and Ph is phenyl optionally substituted with one to five substituents selected from alkyl and aryl groups,
Q2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
A is a chelator residue derived from a chelator selected from the group consisting of 1,4,7,10- tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (=DOTA), N,N'-bis[2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid, l,4,7-triazacyclononane-l,4,7- triacetic acid (=NOTA), 2-(4,7-bis(carboxymethyl)-l,4,7-triazonan-l-yl)pentanedioic acid (=N0DAGA), 2-(4,7, 10-tris(carboxymethyl)- 1 ,4,7, 10-tetraazacyclododecan- 1 - yl)pentanedioic acid (=DOTAGA), 1,4,7-triazacyclononane phosphinic acid (=TRAP), 1,4,7- triazacyclononane-l-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2- hydroxymethyl)phosphinic acid] (=NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca- 1(15), 1 l,13-triene-3,6,9-triacetic acid (=PCTA), l,4-bis(carboxymethyl)-6- [bis(carboxymethyl)]amino-6-methylperhydro-l,4-diazepine (=AAZTA), 1,4,7, 10-tetra- azacyclododecan- 1,4, 7,10-tetra-azidoethylacetic acid (=DOTAZA), Nl-(5-aminopentyl)-Nl- hydroxy-N4-(5-(N-hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4- oxobutanamido)pentyl)succinamide (=DFO), Nl-[5-(Acetylhydroxyamino)pentyl]-N26-(5- aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23 -tetraoxo-5,11, 16,22- tetraazahexacosanediamide (=DFO*), oxo-DFO*, N,N'-bis[(6-carboxy-2-pyridil)methyl]- 4,13-diaza-18-crown-6 (macropa), 4-Amino-6-((16-((6-carboxypyridin-2-yl)methyl)- 1,4,10,13 -tetraoxa-7, 16-diazacyclooctadecan-7-yl)methyl)picolinic acid (=Macropa-NH2)„ 2,2'-(ethane-l,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)azanediyl)) diacetic acid (=H4octox), 6,6'-((9-hydroxy-l,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7- diazabicyclo[3.3.1]nonane-3,7-diyl)bis(methylene))dipicolinic acid (=H2bispa), dimethyl 9- hydroxy-3,7-bis((8-hydroxyquinolin-2-yl)methyl)-2,4-di(pyridin-2-yl)-3,7- diazabicyclo[3.3. l]nonane-l,5-dicarboxylate (=H2bispox), 6,6'-(((pyridine-2,6- diylbis(methylene))-bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid
(=H4py4pa), Diethylenetriaminepentaacetic acid (=DTPA), Trans-cyclohexyl- diethylenetriaminepentaacetic acid (=CHX-DTPA), 1,4,7, 10-tetraazacyclododecane- 1,4, 7- triacetic acid (=D03A), l-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (=oxo-Do3A), 1,4,7, lO-Tetrakis(carbamoylmethyl)- 1,4, 7, 10-tetraazacyclododecane (=D0TAM), N,N'-bis(6- carboxy-2-pyridylmethyl)ethylenediamine-N,N'-diacetic acid (=H4OCtapa), 1,4,7,10,13,16- hexaazacyclohexadecane-l,4,7,10,13,16-hexaacetic acid (=HEHA), 1,4,7,10,13- pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (=PEPA), cyclam-based ligands with N-methyl, N-(4-aminobenzyl), N-phosphinate- and phosphonate-based substituents.
Further, the present invention relates to a complex comprising
(a) a radionuclide, and
(b) a compound, as described above or below, or a pharmaceutically acceptable salt or solvate thereof.
Further, the present invention relates to a pharmaceutical composition comprising a compound, as described above or below, or a pharmaceutically acceptable salt or solvate thereof, 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 pharmaceutically acceptable salt or solvate thereof, or a complex, as described above or below, or a pharmaceutical composition as described above or below, for use in treating or preventing PSMA-expressing cancers, in particular prostate cancer, and/or metastases thereof.
Further, the present invention relates to a method for preparing a compound of formula (1)
Figure imgf000006_0001
or a pharmaceutically acceptable salt or solvate thereof, as described above, the method comprising:
(aa) attaching Fmoc-LysfNPG1 )-OH to a suitable resin, preferably a 2-chloro-trityl resin
(2-CT), and removing the Fmoc protecting group, wherein PG1 is a suitable protecting group, preferably being orthogonal to Fmoc and 2-CT linker of the resin, more preferably Alloc, (bb) reacting H-G1U(OPG2)-OPG2 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming the activated Glu compound of formula (II)
Figure imgf000007_0001
wherein PG2 is a suitable protecting group, preferably being orthogonal to Fmoc and Alloc, more preferably tBu
(cc) reacting the compound of formula (II) with the Lys modified resin according to step
(aa) and removing PG1, thereby forming the modified resin
Figure imgf000007_0004
(dd) reacting the modified resin according to (cc) with Fmoc-(XAA1)-OH, and removing the Fmoc group thereby forming the modified resin
Figure imgf000007_0002
(ee) reacting the modified resin according to (dd) with
Figure imgf000007_0003
and removing the Fmoc group, (ff) linking the free ammo group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L,
(gg) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
Further, the present invention relates to a method for preparing a compound of formula (1)
Figure imgf000008_0001
or a pharmaceutically acceptable salt or solvate thereof, as described above, the method comprising:
(a) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(b) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000008_0002
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu,
(c) reacting the compound of formula (II) with the Glu modified resin according to step
(a) and removing the Fmoc group, thereby forming the modified resin
Figure imgf000009_0001
preferably
Figure imgf000009_0002
(d) reacting the modified resin according to (c) with Fmoc-(XAA1)-OH, and removing the Fmoc group thereby forming the modified resin
H
I 1
Xaa
Figure imgf000009_0003
(e) reacting the modified resin according to (d) with
Fmoc — NH— CH2— Q2 1 OH and removing the Fmoc group,
(f) linking the free amino group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L,
(g) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
Further, the present invention relates to a method for preparing a resin bound PSMA binding moiety having the structure
Figure imgf000010_0001
preferably having the structure
Figure imgf000010_0002
as intermediate product for the solid phase synthesis of PSMA ligands, preferably for the synthesis of a PSMA ligand according to embodiment 1 to 11, the method comprising the steps,
(a) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(b) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000011_0001
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu,
(c) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin.
XAA1
XAA1 is an amino acid moiety having the structure (A)
Figure imgf000011_0002
with Q1 being a residue comprising an optionally substituted styryl moiety, said residue preferably having the structure -(CHR5)m-CH=CH-Ph, where m equals 0, 1, 2, or 3, each R5, independently of others, is selected from the group consisting of H, alkyl and aryl, and Ph is phenyl optionally substituted with one to five substituents selected from alkyl and aryl groups. Preferably, n is 1, 2 or 3, more preferably 1.
Thus, the compound of formula (I), preferably has the structure (la),
Figure imgf000011_0003
wherein R5, R6, R7, R8, R9, and R10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
According to the invention all natural or non-natural amino acids having a chiral C-atoms shall have D- and/or L-configuration; also combinations within one compound shall be possible, i.e. some of the chiral C-atoms may be D- and others may be L-configuration.
Most preferably, all amino acid residues present in the compound have L-configuration.
In particular, XAA1 preferably has L-configuration, compound (I) thus having the structure (laa)
Figure imgf000012_0001
wherein R5, R6, R7, R8, R9, and R10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
The compound of the invention has preferably the structure (lb),
Figure imgf000012_0002
more preferably, the structure (Ibb)
Figure imgf000013_0001
R1, R2, R3 andR4
R1 is H or -CH3, preferably H.
R2, R3 and R4 are independently of each other, selected from the group consisting of -CO2H, - SO2H, -SO3H, -OSO3H, -PO2H2, -PO3H2, -OPO3H2, -CONH2 and -CONHOH. More preferably, R2, R3 and R4 are CO2H.
It is to be understood that carbon atoms present in the urea backbone of the compound if the present invention may be present in any stereoisomeric form, preferably, however, the compounds have the following structures:
Figure imgf000013_0002
preferably
Figure imgf000014_0001
wherein R5, R6, R7, R8, R9, and R10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl. More preferably, the compound of the invention has the structure (Ib-1),
Figure imgf000014_0002
more preferably, the structure (Ibb-1)
Figure imgf000014_0003
(lbb-1), wherein R5, R6, R7, R8, R9, and R10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
Q2
As described above, Q2 is preferably selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl.
The term "alkyl”, as used in this context of the invention, refers to a saturated acyclic hydrocarbon moiety comprising 1 to 10 carbon atoms, preferably 1-6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms, preferably 1 carbon atom.
The term "aryl”, as used in this context of the invention refers to optionally substituted 6- membered aromatic ring, i.e. optionally substituted phenyl group.
The term “alkylaryl” as used in this context of the invention refers to aryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-aryl-) and which are linked via the alkyl group to the -CH2- group and via the aryl group to the carbonyl group.
The term “arylalkyl” as used in this context of the invention refers to aryl groups linked via an alkyl group to the carbonyl group and via the aryl group to the -CH2- group (-aryl-alkyl-).
The term "heteroaryl” (-heteroaryl-), as used in this context of the invention, means optionally substituted, 5- and 6-membered aromatic rings, containing one or more, for example 1 to 4, such as 1, 2, 3, or 4, heteroatoms in the ring system. If more than one heteroatom is present in the ring system, the at least two heteroatoms that are present can be identical or different. Suitable heteroaryl groups are known to the skilled person. The following heteroaryl residues may be mentioned, as non-limiting examples: pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, and pyridazinyl.
The term “alkylheteroaryl” as used in this context of the invention refers to heteroaryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-heteroaryl-) and which are linked via the alkyl group to the -CH2- group and via the heteroaryl group to the carbonyl group.
The term “heteroarylalkyl” as used in this context of the invention refers to heteroaryl groups linked via the alkyl group to the carbonyl group and via the heteroaryl group to the -CH2- group (-heteroaryl-alkyl-).
The term “cycloalkyl” (-cycloalkyl-) means, in the context of the invention, optionally substituted, 4-10-membered cyclic alkyl residues, wherein they can be monocyclic or polycyclic groups. Optionally substituted cyclohexyl may be mentioned as a preferred example of a cycloalkyl residue. The term "heterocycloalkyl", as used in this context of the invention refers to optionally substituted, 4-10-membered cyclic alkyl residues, which have at least one heteroatom, such as O, N or S in the ring, wherein they can be monocyclic or polycyclic groups.
The terms "substituted ", as used in this context of the invention, refer to residues, in which at least one H has been replaced with a suitable substituent.
Preferably, Q2 is an optionally substituted aryl group or optionally substituted cycloalkyl group, in particular optionally substituted phenyl or optionally substituted cyclohexyl.
Thus, Q2 is preferably
Figure imgf000016_0003
wherein R11, R12, R13 and R14 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl.
More preferably Q2 is
Figure imgf000016_0001
It is to be understood that any stereoisomers of Q2 are possible and included. In case Q2 is
Figure imgf000016_0002
it is to be understood that this includes the cis as well as the trans isomer, with the trans isomer being particularly preferred.
Thus, the present invention also preferably also relates to compounds having the structure
Figure imgf000017_0001
wherein R5, R6, R7, R8, R9, and R10 are, independently of each other, selected from the group consisting of H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl, and wherein R11, R12, R13 and R14 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl. More preferably, the compound of the invention has the structure,
Figure imgf000018_0001
Chelator residue A
A is a chelator residue derived from a chelator selected from the group consisting of 1,4,7,10- tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (=DOTA), N,N'-bis[2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid, l,4,7-triazacyclononane-l,4,7- triacetic acid (=NOTA), 2-(4,7-bis(carboxymethyl)-l,4,7-triazonan-l-yl)pentanedioic acid (=N0DAGA), 2-(4,7, 10-tris(carboxymethyl)- 1 ,4,7, 10-tetraazacyclododecan- 1 - yl)pentanedioic acid (=DOTAGA), 1,4,7-triazacyclononane phosphinic acid (=TRAP), 1,4,7- triazacyclononane-l-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2- hydroxymethyl)phosphinic acid] (=NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca- 1(15), 1 l,13-triene-3,6,9-triacetic acid (=PCTA), l,4-bis(carboxymethyl)-6-
[bis(carboxymethyl)]amino-6-methylperhydro-l,4-diazepine (=AAZTA), 1,4,7, 10-tetra- azacyclododecan- 1,4, 7,10-tetra-azidoethylacetic acid (=DOTAZA), Nl-(5-aminopentyl)-Nl- hydroxy-N4-(5-(N-hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4- oxobutanamido)pentyl)succinamide (=DFO), Nl-[5-(Acetylhydroxyamino)pentyl]-N26-(5- aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23 -tetraoxo-5,11, 16,22- tetraazahexacosanediamide (=DFO*), oxo-DFO*, N,N'-bis[(6-carboxy-2-pyridil)methyl]- 4,13-diaza-18-crown-6 (macropa), 4-Amino-6-((16-((6-carboxypyridin-2-yl)methyl)- 1,4,10,13 -tetraoxa-7, 16-diazacyclooctadecan-7-yl)methyl)picolinic acid (=Macropa-NH2), 2,2'-(ethane-l,2-diylbis(((8-hydroxyquinolin-2-yl)methyl)azanediyl)) diacetic acid (=H4octox), 6,6'-((9-hydroxy-l,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7- diazabicyclo[3.3.1]nonane-3,7-diyl)bis(methylene))dipicolinic acid (=H2bispa), dimethyl 9- hydroxy-3,7-bis((8-hydroxyquinolin-2-yl)methyl)-2,4-di(pyridin-2-yl)-3,7- diazabicyclo[3.3. l]nonane-l,5-dicarboxylate (=H2bispox), 6,6'-(((pyridine-2,6- diylbis(methylene))-bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid
(=H4py4pa), Diethylenetriaminepentaacetic acid (=DTPA), Trans-cyclohexyl- diethylenetriaminepentaacetic acid (=CHX-DTPA), 1,4,7, 10-tetraazacyclododecane- 1,4, 7- triacetic acid (=D03A), l-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (=oxo-Do3A), 1,4,7, lO-Tetrakis(carbamoylmethyl)- 1,4, 7, 10-tetraazacyclododecane (=DOTAM), N,N'-bis(6- carboxy-2-pyridylmethyl)ethylenediamine-N,N'-diacetic acid (=H4OCtapa), 1,4,7,10,13,16- hexaazacyclohexadecane-l,4,7,10,13,16-hexaacetic acid (=HEHA), 1,4,7,10,13- pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (=PEPA), cyclam-based ligands with N-methyl, N-(4-aminobenzyl), N-phosphinate- and phosphonate-based substituents.
The chelator oxo-DFO* has the structure:
Figure imgf000019_0001
Reference is made to Manon Briand, Margaret L Aulsebrook, Thomas L Mindt, Gilles Gasser, Dalton Transactions, 2017, 46 (47), pp.16387-16389 which contents regarding oxo-DFO* is herewith enclosed by reference.
The term “a chelator residue” and typically also the term “chelator residue derived from a chelator selected from the group” is denoted to mean that the above mentioned chelators, thus typically the chelators defined in the “group”, have been linked, via a suitable group to the group -(L)n-NH-, i.e. to the residue
Figure imgf000019_0002
thereby forming a covalent bond between the chelator and the compound (I).
The linking moiety (L)n The linking moiety (L)n is chosen, depending on the nature of the chelator residue A and the functional groups capable of being linked to the rest of the compound being present in A.
Preferably L is a linking group selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q- C(=O)-, -(Q3)q-C(=S)- and -(Q3)q-NH-C(=S)-, wherein Q3 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is 0 or 1.
The term "alkyl”, as used in this context of the invention, refers to a saturated acyclic hydrocarbon moiety comprising 1 to 10 carbon atoms, preferably 1-6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms, preferably 1 carbon atom.
The term "aryl”, as used in this context of the invention refers to optionally substituted 6- membered aromatic ring, i.e. optionally substituted phenyl group.
The term “alkylaryl” as used in this context of the invention refers to aryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-aryl-) and which are linked via the alkyl group to A and via the aryl group to the rest of linker L.
The term “arylalkyl” as used in this context of the invention refers to aryl groups linked via an alkyl group to the rest of linker L and via the aryl group to A (-aryl-alkyl-).
The term "heteroaryl” (-heteroaryl-), as used in this context of the invention, means optionally substituted, 5- and 6-membered aromatic rings, containing one or more, for example 1 to 4, such as 1, 2, 3, or 4, heteroatoms in the ring system. If more than one heteroatom is present in the ring system, the at least two heteroatoms that are present can be identical or different. Suitable heteroaryl groups are known to the skilled person. The following heteroaryl residues may be mentioned, as non-limiting examples: pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyridazinyl, and pyridazinyl.
The term “alkylheteroaryl” as used in this context of the invention refers to heteroaryl groups in which at least one proton has been replaced with an alkyl group (-alkyl-heteroaryl-) and which are linked via the alkyl group to A and via the heteroaryl group to the rest of linker L.
The term “heteroarylalkyl” as used in this context of the invention refers to heteroaryl groups linked via the alkyl group to the rest of linker L and via the heteroaryl group to A group (- heteroaryl-alkyl-).
The terms "substituted", as used in this context of the invention, refer to residues, in which at least one H has been replaced with a suitable substituent.
In case A is linked via a Carboxy group present in A to the rest of the compound, n is preferably 0 and the Carboxy group is directly linked to the Amino group adjacent to group CH2-Q2-
In case A is linked via amino group present in A to the rest of the molecule, n is preferably 1 and L is preferably selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q-C(=O)-, - (Q3)q-C(=S)- and -(Q3)q-NH-C(=S)-, wherein Q3 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, more preferably wherein L is -(Q3)q-C(=O)- or -(Q3)q-C(=S)-, in particular, wherein L is -(Q3)q-C(=O)-, -(Q3)q- C(=S)-and wherein q is 0, i.e. L is -C(=O)- or -C(=S)-.
Alternatively, A is linked to the rest of the molecule via L, with n being 1 and with L being selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q-C(=O)-, -(Q3)q-C(=S)- and (Q3)q-NH-C(=S), in particular wherein L is selected from the group consisting of -alkyl-NH C(=O)-, -aryl-NH-C(=O)-, -alkylaryl-NH-C(=O)-, -arylalkyl-NH-C(=O)-, -heteroaryl-NH C(=O)-, -heteroarylalkyl-NH-C(=O)-, -alkylheteroaryl-NH-C(=O)-, -alkyl-C(=O)-, -aryl C(=O)-, -alkylaryl-C(=O)-, -arylalkyl-C(=O)-, -heteroaryl-C(=O)-, -heteroarylalkyl-C(=O)-, alkylheteroaryl-C(=O)-, -alkyl-C(=S)-, -aryl-C(=S)-, -alkylaryl-C(=S)-, -arylalkyl-C(=S)-, heteroaryl-C(=S)-, -heteroarylalkyl-C(=S)-, -alkylheteroaryl-C(=S)-, alkyl-NH-C(=S)-, -aryl NH-C(=S)-, -alkylaryl-NH-C(=S)-, -arylalkyl-NH-C(=S)-, -heteroaryl-NH-C(=S)-, heteroarylalkyl-NH-C(=S)-, and -alkylheteroaryl-NH-C(=S)-, preferably with L being (alkylaryl)-NH-C(=O)- or -(alkylaryl)-NH-C(=S)-, more preferably -(benzyl)-NH-C(=O)- or (benzyl)-NH-C(=S)-.
According to an alternative embodiment, n is i and L is -NH-C(=O)- or -NH-C(=S)-.
Preferably, A is a chelator residue selected from the group consisting of
Figure imgf000021_0001
N,N'-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6 (macropa), 4-Amino-6-((16- ((6-carboxypyridin-2-yl)methyl)-l,4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7- yl)methyl)picolinic acid (=Macropa-NH2), and 1,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (=HEHA).
More preferably, A is a chelator residue selected from the group consisting of
Figure imgf000021_0002
Figure imgf000022_0001
In case A is a chelator residue having a structure selected from the group consisting of
Figure imgf000022_0002
n is preferably 0.
In case A is a chelator residue having a structure
Figure imgf000023_0001
n is preferably 1 and L is preferably -(benzyl)-NH-C(=O)- or -(benzyl)-NH-C(=S)-
In case A is a chelator residue having a structure
Figure imgf000023_0002
n is preferably 1 and L is preferably -C(=O)- or -C(=S)-.
More preferably, A is
Figure imgf000023_0003
In this case, n is preferably 0. More preferably, the compound has the structure Pl 8
Figure imgf000024_0001
or the structure P17
Figure imgf000024_0002
most preferably, the structure Pl 7.
According to a further preferred embodiment, A is a chelator residue having a structure
Figure imgf000025_0001
and n is 1 and L is -(benzyl)-NH-C(=O)- or -(benzyl)-NH-C(=S)-, more preferably, the compound has the structure
Figure imgf000025_0002
According to a further preferred embodiment, A is a chelator residue having a structure
Figure imgf000026_0001
and nis i and L is -C(=0)- or -C(=S)-, more preferably, the compound has the structure
Figure imgf000026_0002
5
Figure imgf000027_0001
Complex
As described above, the present invention also relates to a complex comprising
(a) a radionuclide, and
(b) a compound, as described above or below, or a pharmaceutically acceptable salt or solvate thereof.
Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as /?-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenyl sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bi sulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1,4- dioate, hexyne- 1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, napththalene-2-sulfonate, mandelate and the like. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid. Salts of amine groups may also comprise quaternary ammonium salts in which the amino nitrogen carries a suitable organic group such as an alkyl, alkenyl, alkynyl, or aralkyl moiety. Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like. The potassium and sodium salt forms are particularly preferred. It should be recognized that the particular counter ion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counter ion does not contribute undesired qualities to the salt as a whole.
The term “pharmaceutically acceptable solvate” encompasses also suitable solvates of the compounds of the invention, wherein the compound combines with a solvent such as water, methanol, ethanol, DMSO, acetonitrile or a mixture thereof to form a suitable solvate such as the corresponding hydrate, methanolate, ethanolate, DMSO solvate or acetonitrilate.
The radionuclide
The complexes of invention may contain one or more radionuclides, preferably one radionuclide. These radionuclides are preferably suitable for use as radio-imaging agents or as therapeutics for the treatment of proliferating cells, for example, PSMA expressing cancer cells, in particular PSMA-expressing prostate cancer cells. According to the present invention they are called "radionuclide complexes" or "radiopharmaceuticals".
Preferred imaging methods are positron emission tomography (PET) or single photon emission computed tomography (SPECT).
Preferably, the at least one radionuclide is selected from the group consisting 18F, 89Zr, 43Sc, 44Sc, 47Sc, 45Ti, 55Co, mIn, 86Y, 90Y, 66Ga, 67Ga, 68Ga, 177Lu, 99mTc, 60Cu, 61Cu, 62Cu, 64Cu, 66Cu, 67Cu, 149Tb, 152Tb, 155Tb, 153Sm, 161Tb, 166Ho, 153Gd, 155Gd, 157Gd, 211At, 213Bi, 225 Ac, 230U, 223Ra, 224Ra, 165Er, 52Fe, 59Fe, 186Re, 188Re, 227Th, 133La, 72 As and radionuclides of Pb, such as 203Pb and 212Pb.
More preferably, the at least one radionuclide is selected from the group consisting 18F, 68Ga, 133La, 177LU, 188Re, and 225 Ac. More preferably, the radionuclide is 18F, 68Ga, 177Lu, 188Re, and 225 Ac.
Preferably, the radionuclide has a half-life of at least 30 min, more preferably of at least 1 h, more preferably at least 12 h, even more preferably at least Id, most preferably at least 5 d; also preferably, the radionuclide has a half-life of at most 1 year, more preferably at most 6 months, still more preferably at most 1 month, even more preferably at most 14 d. Thus, preferably, the radionuclide has a half-life of from 30 min to 1 year, more preferably of 12 h to 6 months, even more preferably of from 1 d to 1 month, most preferably of from 5 d to 14 d. The radionuclide according to the invention may be an a-emitter, a P -emitter, a P+-emitter, a y-emitter, and/or mixed-emitter from all options mentioned before.
Preferably, the radionuclide is an a- and/or P-emitter, i.e. the radionuclide preferably emits a- particles (a-emitter) and/or P-radiation (P-emitter).
Preferably, in case the radionuclide is an a-emitter, the a-particle has an energy of from 1 to 10 MeV, more preferably of from 2 to 8 MeV, most preferably of from 4 to 7 MeV.
Preferably, in case the radionuclide is a P-emitter, the P-radiation has an energy of from 0.1 to 10 MeV, more preferably of from 0.25 to 5 MeV, most preferably of from 0.4 to 2 MeV.
Preferred radionuclides emitting P-radiation are selected from the group consisting of90Y, 177Lu and 188Re.
Preferred radionuclides emitting a -radiation are e.g. selected from the group consisting of 213Bi, 223Ra, 225 Ac and 227Th.
According to a further embodiment, the radionuclide is a positron emitter. In this case the radionuclide is preferably selected from the group consisting of 18F, 44Sc, 55Co, 64Cu, 66Ga, 68Ga, 72 As and 89Zr. In this case, the use is preferably PET diagnosis.
According to a further preferred embodiment, radionuclide is a gamma emitter. In this case the radionuclide is preferably U 1ln or 177Lu. In this case, the use preferably is SPECT diagnosis.
According to a further preferred embodiment, the radionuclide emits Auger electrons, and preferably decays by electron capture. In this case, the radionuclide is preferably 161Tb. In this case, the use is preferably therapy.
Method 1
As describe above and below, the present invention also relates to a method for preparing a compound of formula (1) or a pharmaceutically acceptable salt or solvate thereof with compound (I) being as describe above and below,
Figure imgf000029_0001
the method comprising:
(aa) attaching Fmoc-LysfNPG1 )-OH to a suitable resin, preferably a 2-chloro-trityl resin
(2-CT), and removing the Fmoc protecting group, wherein PG1 is a suitable protecting group, preferably being orthogonal to Fmoc and 2-CT linker of the resin, more preferably Alloc,
(bb) reacting H-G1U(OPG2)-OPG2 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming the activated Glu compound of formula (II)
Figure imgf000030_0001
wherein PG2 is a suitable protecting group, preferably being orthogonal to Fmoc and Alloc, more preferably tBu
(cc) reacting the compound of formula (II) with the Lys modified resin according to step
(aa) and removing PG1, thereby forming the modified resin
Figure imgf000030_0002
(dd) reacting the modified resin according to (cc) with Fmoc-(XAA1)-OH, and removing the Fmoc group thereby forming the modified resin
Figure imgf000030_0003
reacting the modified resin according to (dd) with
Figure imgf000030_0004
and removing the Fmoc group,
(ff) linking the free amino group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L,
(gg) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
Step (aa)
In step (aa) Fmoc-Lys(NPG' )-OH is attached to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), the Fmoc protecting group is removed, wherein PG1 is a suitable protecting group, preferably being orthogonal to Fmoc and 2-CT linker of the resin.
Preferably step (aa) comprises
(aal) attaching Fmoc-Lys(NPG1)-OH to the resin, preferably the 2-chloro-trityl resin in the presence of a base
(aa2) removing the Fmoc group.
Step (aal) is preferably carried out in the presence of a suitable base. Such bases are known to the skilled person and include, in particular, organic bases, more preferably amino group comprising bases. Preferably, the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N- methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4- dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (aa) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
Preferably, step (aal) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofuran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), di chloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
Methods to remove Fmoc groups are known to the skilled person. Preferably step (aa2) is carried out in the presence of piperidine in DMF as solvent.
Thus, step (aa) in particular comprises
(aal) attaching Fmoc-Lys(NPG1)-OH to the resin, preferably the 2-chloro-trityl resin in the presence of A,A-ethyldiisopropylamine (DIPEA), and
(aa2) removing the Fmoc group in the presence of piperidine in DMF. The protecting group PG1 is preferably selected from the group consisting of acetyl, trifluoroacetyl, DDE (l-(4,4-Dimethyl-2,6-dioxocyclohex-l-ylidene)-3-eth-yl), ivDDE ( 1- (4,4-Dimethyl-2,6-dioxocyclohex-l-ylidene)-3-methylbutyl) , CbZ (N-carboxybenzyl), Benzyl, Tosyl, Phtalimide, more preferably Alloc.
Step (bb)
In step (bb), H-G1U(OPG2)-OPG2 is reacted with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming the activated Glu compound of formula (II)
Figure imgf000032_0001
wherein PG2 is a suitable protecting group, preferably being orthogonal to Fmoc and Alloc, more preferably tBu
Preferably, the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N- methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N- methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4-dimethylaminopyridine, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (bb) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
Preferably, step (bb) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofiiran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), di chloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
Step (cc)
In step (cc), the compound of formula (II), preferably the compound of formula (Ila), is reacted the with the Lys modified resin according to step (aa) and PG1 is removed, thereby forming the modified resin
Figure imgf000033_0001
Thus step (cc) preferably comprises
(ccl) the compound of formula (Ila), is reacted the with the Lys modified resin according to step (aa), and
(cc2) removing PG1.
Preferably, PG1 is an Alloc protecting group and the group is removed by reaction with TPP palladium in the presence of morpholine.
Preferably step (ccl) is carried out in the presence of a suitabl base, such as piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N- methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4- dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (ccl) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
Step (dd)
In step (dd) the modified resin according to (cc) is reacted with Fmoc-(XAA1)-OH, and Fmoc group is removed thereby forming the modified resin
Figure imgf000033_0002
Thus, step (dd) preferably comprises
(ddl) reacting the modified resin according to (cc) with Fmoc-(XAA1)-OH,
(dd2) removing the Fmoc group thereby forming the modified resin.
Figure imgf000034_0002
Preferably step (ddl) is carried out in the presence of a suitable activation reagent. Such coupling reagents include, but are not limited to, HATU (O-(7-azabenzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate); HOAt, HBTU (O-benzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate); TBTU (2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate); TFFH (N,N',N",N"-tetramethyluronium-2-fluoro- hexafluorophosphate); BOP (benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate); PyBOP (benzotriazol- 1 -yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate; EEDQ (2-ethoxy-l-ethoxycarbonyl-l,2-dihydro-quinoline); DCC (dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide); HOBt (1- hydroxybenzotriazole); NHS (N-hydroxy succinimide); MSNT (l-(mesitylene-2-sulfonyl)-3- nitro-lH-l,2,4-triazole); aryl sulfonyl halides, e.g. triisopropylbenzenesulfonyl chloride, EDC (l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride, CDC (l-cyclohexyl-3-(2- morpholinoethyl)carbodiimide), Pyclop, T3P, CDI, Mukayama’s reagent, HODhbt, HAPyU, TAPipU, TPTU, TSTU, TNTU, TOTU, BroP, PyBroP, BOI, TOO, NEPIS, BBC, BDMP, BOMI, AOP, BDP, PyAOP, TDBTU, BOP-CI, CIP, DEPBT, Dpp-Cl, EEDQ, FDPP, HOTT, TOTT, PyCloP, and Oxyma Pure (Ethyl cyano(hydroxyimino)acetate) in combination with DIC, more preferably HBTU or Oxyma Pure and DIC.
Methods to remove Fmoc groups are known to the skilled person. Preferably step (dd2) is carried out in the presence of piperidine in DMF as solvent.
Step (ee)
In step (ee), the modified resin according to (dd) is reacted with Fmoc-
Figure imgf000034_0001
and the Fmoc group is removed.
Thus, step (ee) preferably comprises (eel) reacting the modified resin according to (dd) with Fmoc-
Figure imgf000035_0001
(ee2) removing the Fmoc group.
The reaction in (ee) is preferably carried out similar to the reaction in (ddl), thus in the presence of a suitable activation reagent, Reference is made to the activation reagents mention in the context of (ddl) which are also preferred in (eel).
Step (ff)
In step (ff), the free amino group is linked with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L. Thereby, preferably, the group A-(L)n-NH- is formed.
If A is linked via a carboxy group present in A or in L, to the NH group, the reaction may be carried out in the presence of a suitable activation reagent, such as the reagents mentioned in the context of step (eel). In this case, in step (ff) A-(L)n is preferably provided in the form of an NHS ester and is subsequently attached to free amino group. Thereby, preferably, the group A-(L)n-NH- is formed which is more preferably A-(Q3)q-C(=O)-NH-, with Q3 and q being as described above and below.
Alternatively, A-(L)n- moiety may be linked via an amine group. In this case, A-(L)n may be provided in form of an activated isothiocyanate with which the free amino group may be reacted. In this case, n is preferably 1 and L is provided in the form -(Q3)q-NCS, with Q3 and q being as described above and below. Upon reaction with the free amino group, preferably the group A-(L)n- NH-, more preferably A-(Q3)q-NH-C(=S)-NH- is formed.
Step (gg)
In step (gg), the compound is cleaved from the resin and all remaining protecting groups are removed. Suitable cleaving conditions are known to the skilled person. Preferably, step (gg) is carried out in the presence of trifluoroacidic acid.
Method 2:
As describe above and below, the present invention preferably relates to a method for preparing a resin bound PSMA binding moiety having the structure
Figure imgf000035_0002
preferably
Figure imgf000036_0001
more preferably
Figure imgf000036_0002
more preferably
Figure imgf000036_0003
as intermediate product for the solid phase synthesis of PSMA ligands, preferably for the synthesis of a PSMA ligand of as describe above and below, the method comprising the steps,
(a) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-Chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(b) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000037_0001
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu,
(c) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin.
Step (a)
In step Fmoc-Glu(OPG2)-OH is attached to a suitable resin, preferably a 2-Chloro-trityl resin (2-CT), and the Fmoc protecting group is removed, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
Preferably step (a) comprises
(al) attaching Fmoc-Glu(OPG2)-OH to the resin, preferably the 2-chloro-trityl resin in the presence of a base
(a2) removing the Fmoc group.
Step (al) is preferably carried out in the presence of a suitable base. Such bases are known to the skilled person and include, in particular, organic bases, more preferably amino group comprising bases. Preferably, the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), 7V,7V-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N- methylmorpholine, N-methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N- methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4- dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (aa) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
Preferably, step (al) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofuran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), di chloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
Methods to remove Fmoc groups are known to the skilled person. Preferably step (a2) is carried out in the presence of piperidine in DMF as solvent.
The protecting group PG2 is preferably selected from the group consisting of methylester, t- butylester, benzylester, S-t-butylester, more preferably tBu. Step (b)
Reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000038_0001
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu.
Preferably, the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), 7V,7V-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N- methylimidazole, l,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N- methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4-dimethylaminopyridine, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (bb) is carried out in the presence of A,A-ethyldiisopropylamine (DIPEA).
Preferably, step (b) is carried out in an organic solvent, such as N-methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), acetonitrile, acetone, dimethyl acetamide (DMA), dimethyl formamide (DMF), formamide, tetrahydrofuran (THF), 1,4-di oxane, diethyl ether, tert. -butyl methyl ether (MTBE), dichloromethane (DCM), chloroform, tetrachloromethane and mixtures of two or more thereof. More preferably, the reaction is carried out in dichloromethane.
Step (c)
In step (c) the compound of formula (II) is reacted with the Glu modified resin according to step (a) and the Fmoc group is removed, thereby forming the modified resin having the structure
Figure imgf000038_0002
preferably
Figure imgf000039_0001
more preferably
Figure imgf000039_0002
more preferably
Figure imgf000039_0003
Thus, step (c), preferably comprises
(cl) the compound of formula (Ila), is reacted the with the Glu modified resin according to step (a), and
(c2) removing the Fmoc group.
Preferably, (cl) is carried out in the presence of a base, wherein the base is selected from the group consisting of piperidine, diisopropylamine (DIEA), A,A-ethyldiisopropylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N-methylimidazole, 1,4- diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, collidine, pyridine, 4-dimethylaminopyridine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and mixtures thereof. More preferably, step (bb) is carried out in the presence of N,N- ethyldiisopropylamine (DIPEA).
The resin bound PSMA binding moiety obtained by the method comprising steps (a) to (c) having the structure
Figure imgf000040_0001
preferably
Figure imgf000040_0002
more preferably
Figure imgf000040_0003
more preferably
Figure imgf000040_0004
is preferably used as intermediate product for the preparation of the PSMA ligands according to the invention described above and below.
The method for the preparation of the intermediate product is particularly advantageous since no hazardous reagents, such as triphosgene, is used. Further, there is no need to use an Alloc protecting group for the deprotection of which hazardous TPP palladium in the presence of morpholine needs to be used. Preferably, the present invention thus also relates to a method for preparing a compound of formula (1) in which the intermediate compound is prepared according to steps (a) to (c) described above and which is then transformed in further reaction steps to a PSMA ligand according to the invention.
Thus, the present invention also relates to method for preparing a compound of formula (1)
(a) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(b) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000041_0001
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu, (c) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin
Figure imgf000041_0002
preferably
Figure imgf000041_0003
more preferably
Figure imgf000042_0001
more preferably
Figure imgf000042_0002
(d) reacting the modified resin according to (c) with Fmoc-(XAAi)-OH, and removing the Fmoc group thereby forming the modified resin
Figure imgf000042_0003
more preferably
Figure imgf000043_0001
more preferably
Figure imgf000043_0002
and removing the Fmoc group,
(f) linking the free amino group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L,
(g) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
Step (e)
In step (e) the modified resin according to (d) is reacted with Fmoc
Figure imgf000044_0001
and the Fmoc group is removed.
Thus, step (e) preferably comprises
(el) reacting the modified resin according to (d) with
Fmoc
Figure imgf000044_0002
(e2) removing the Fmoc group.
The reaction in (e) is preferably carried out similar to in the presence of a suitable activation reagent, Reference is made to the activation reagents mention in the context of (ddl) which are also preferred in (el).
Preferably, step (el) is carried out in the presence of HBTU or Oxyma Pure and DIC.
Methods to remove Fmoc groups are known to the skilled person. Preferably step (e2) is carried out in the presence of piperidine in DMF as solvent.
Step (f)
If A is linked via a carboxy group present in A or in L, to the NH group, the reaction may be carried out in the presence of a suitable activation reagent, such as the reagents mentioned in the context of step (el). In this case, in step (ff) A-(L)n is preferably provided in the form of an NHS ester and is subsequently attached to free amino group. Thereby, preferably, the group A-(L)n-NH- is formed which is more preferably A-(Q3)q-C(=O)-NH-, with Q3 and q being as described above and below.
Alternatively, A-(L)n- moiety may be linked via an amine group. In this case, A-(L)n may be provided in form of an activated isothiocyanate with which the free amino group may be reacted. In this case, n is preferably 1 and L is provided in the form -(Q3)q-NCS, with Q3 and q being as described above and below. Upon reaction with the free amino group, preferably the group A-(L)n-NH-, more preferably A-(Q3)q-NH-C(=S)-NH- is formed.
Step (g)
In step (g), the compound is cleaved from the resin and all remaining protecting groups are removed. Suitable cleaving conditions are known to the skilled person. Preferably, step (g) is carried out in the presence of trifluoroacidic acid.
The present invention also relates to a compound of formula (1), obtained or obtainable by a method according described herein. Pharmaceutical compositions and therapeutic uses
Also, the present invention relates to a pharmaceutical composition comprising a compound or a complex as specified herein. Further, the present invention relates to a compound, a complex, or a pharmaceutical composition as described herein for use in medicine, in particular for use in treating and/or preventing a PSMA expressing cancer, in particular prostate cancer and/or metastases thereof. The present invention also relates to a method for treating a PSMA expressing cancer in a patient, the method comprising (A) contacting said patient with a compound, a complex, or a pharmaceutical composition as described herein to said sample; and (B) thereby treating said cancer in said patient. The present invention also relates to a use of a compound, a complex, or a pharmaceutical composition as described herein for the manufacture of a medicament for treating a PSMA expressing cancer.
The terms "medicament" and "pharmaceutical composition" are used essentially interchangeably herein and are, in principle, known to the skilled person. As referred to herein, the terms relate to any composition of matter comprising the specified active agent(s) as pharmaceutically active compound(s) and one or more excipient. The pharmaceutically active compound(s) can be present in liquid or dry, e.g. lyophilized, form. It will be appreciated that the form and character of the pharmaceutical acceptable excipient, e.g. carrier or diluent, is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. The excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The excipient employed may include a solid, a gel, or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution, syrup, oil, water, emulsions, various types of wetting agents, and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The excipient(s) is/are selected so as not to affect the biological activity of the combination. The excipient may, however, also be selected to improve uptake of the active agent into a host cell, in particular a target cell.
Preferably, the medicament is, typically, administered systemically, preferably orally or parenterally, e.g. by intravenous administration, or is administered topically, preferably intra- tumorally, topically on a body surface, or by inhalation; in case of cancer treatment, topical administration may be intratumoral or peritumoral, and/or topical at a site of tumor excision. Administration may, however, also be into a blood vessel, typically an artery, afferent to an intended site of effect, such as a tumor. However, depending on the nature of the formulation and the desired therapeutic application, the medicament may be administered by other routes as well. The medicament is preferably administered in conventional dosage forms prepared by combining the drug with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating, and compression, or dissolving the ingredients as appropriate to obtain the desired preparation. A therapeutically effective dose refers to an amount of the active substance to be used in a medicament which prevents, ameliorates or cures the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of a drug can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, age, the particular formulation of the medicament to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The medicament referred to herein is, preferably, administered at least once, e.g. as a bolus. However, the medicament may be administered more than one time and, preferably, at least twice, e.g. permanently or periodically after defined time windows. Progress can be monitored by periodic assessment. Dosage recommendations may be indicated in the prescriber or user instructions in order to anticipate dose adjustments depending on the considered recipient. Typical doses for compounds similar to those of described herein, e.g. PSMA-617, are known in the art, e.g. from WO 2020/165420. In view of the pharmacokinetic properties of the compounds described herein, essentially the same dosages are applicable for these as well.
The medicament according to the present invention may comprise further active agents in addition to the aforementioned active agent(s). Preferably, the pharmaceutically active compound according to the invention is to be applied together with at least one further drug and, thus, may be formulated together with this at least one further drug as a medicament. More preferably, in case of cancer treatment, said at least one further active agent is a chemotherapeutic agent or an immunotherapeutic agent, such as an immune checkpoint modulator. Also, it is to be understood that the formulation of a pharmaceutical composition preferably takes place under GMP standardized conditions or the like in order to ensure quality, pharmaceutical safety, and effectiveness of the medicament.
The term "cancer", as used herein, relates to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells (“cancer cells”). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue and possibly spread of cancer cells to other locations in the body (metastasis). Preferably, also included by the term cancer is a relapse. Thus, preferably, the cancer is a solid cancer, a metastasis, or a relapse thereof. Preferably, the cancer is an uncontrolled proliferation of cells comprising cells expressing PSMA. Also preferably, the cancer is a solid cancer comprising cells expressing PSMA, e.g. cells forming blood vessels, preferably in neovasculature. Thus, the cancer may in particular be a PSMA expressing cancer such as prostate cancer, thyroid cancer, salivary gland cancer, lung cancer, liver cancer, kidney cancer, breast cancer, or brain cancer, or a metastasis of any of the aforesaid. More preferably, the cancer is prostate cancer and/or a metastasis thereof
The term "patient", as used herein, relates to a vertebrate, preferably a mammalian animal, more preferably a human, monkey, cow, horse, cat or dog. Preferably, the mammal is a primate, more preferably a monkey, most preferably a human. Preferably, the patient is known or suspected to suffer from a cancer as specified herein.
The terms "treating" and “treatment” refer to an amelioration of the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of health with respect to the diseases or disorders referred to herein. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, as specified herein above. The term “preventing” and "prevention" refers to retaining health with respect to the diseases or disorders referred to herein for a certain period of time in a subject. It will be understood that the said period of time may be dependent on the amount of the drug compound which has been administered and individual factors of the subject discussed elsewhere in this specification. It is to be understood that prevention may not be effective in all subjects treated with the compound according to the present invention. However, the term requires that, preferably, a statistically significant portion of subjects of a cohort or population are effectively prevented from suffering from a disease or disorder referred to herein or its accompanying symptoms. Preferably, a cohort or population of subjects is envisaged in this context which normally, i.e. without preventive measures according to the present invention, would develop a disease or disorder as referred to herein. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools discussed herein above. Preferably, treatment and/or prevention comprises administration of at least one compound as specified herein and/or at least one complex as specified elsewhere herein.
In view of the above, the invention provides a method for treating a patient suffering from cancer by administering to said patient a therapeutically effective amount of a compound, complex, or medicament, all as described above or below, to treat a patient suffering from a cancer.
Diagnostic uses
The present invention also relates to a compound as described herein, a complex as described herein, or a pharmaceutical composition as described herein, for use in diagnostics, in particular for use in the diagnosis of cancer, preferably of PSMA expressing cancer, in particular of prostate cancer and/or metastases thereof. Said diagnostic methods preferably are practised on the human or animal body. Further, the present invention relates to a compound as described herein, a complex as described herein, or a pharmaceutical composition as described herein for use in diagnosing a PSMA expressing cancer in a patient, said diagnosing comprising (i) determining binding of said compound or complex to a tissue of said subject; (ii) comparing the binding determined in step (i) to a reference; (iii) identifying a difference in step (ii); and (iv) diagnosing said cancer based on the result of step (iii). The present invention also relates to a method for diagnosing a PSMA expressing cancer in a sample of a patient, the method comprising (I) determining binding of a compound, a complex, or a pharmaceutical composition as described herein to said sample; and (II) diagnosing said cancer based on the result of step (I); said method may comprise further step (la) comparing the binding determined in step (I) to a reference; in such case, step (II) preferably comprises diagnosing said cancer based on the result of step (la), more preferably is step (Ila) diagnosing said cancer based on the result of step (la). The present invention also relates to a use of a compound, a complex, or a pharmaceutical composition as described herein for the manufacture of a diagnostic for diagnosing a PSMA expressing cancer.
The term “diagnosing”, as used herein, refers to assessing whether a subject suffers from a disease or disorder, preferably cell proliferative disease or disorder, or not. As will be understood by those skilled in the art, such an assessment, although preferred to be, may usually not be correct for 100% of the investigated subjects. The term, however, requires that a, preferably statistically significant, portion of subjects can be correctly assessed and, thus, diagnosed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The p-values are, preferably, 0.2, 0.1, or 0.05. As will be understood by the skilled person, diagnosing may comprise further diagnostic assessments, such as visual and/or manual inspection, determination of tumor biomarker concentrations in a sample of the subject, X-ray examination, and the like. The term includes individual diagnosis of as well as continuous monitoring of a patient. Monitoring, i.e. diagnosing the presence or absence of cell proliferative disease or the symptoms accompanying it at various time points, includes monitoring of patients known to suffer from cancer as well as monitoring of subjects known to be at risk of developing cell proliferative disease. Furthermore, monitoring can also be used to determine whether a patient is treated successfully or whether at least symptoms of cell proliferative disease can be ameliorated over time by a certain therapy. Moreover, the term also includes classifying a subject according to a usual classification scheme, e.g. the T1 to T4 staging, which is known to the skilled person.
Preferably the compound, as described above or below, or the complex, as described above or below, or the pharmaceutical composition, as described above or below, are used for in vivo imaging and radiotherapy. Suitable pharmaceutical compositions may contain a radio imaging agent, or a radiotherapeutic agent that has a radionuclide either as an element, i.e. radioactive iodine, or a radioactive metal chelate complex of the compound of formula (la) and/or (lb) in an amount sufficient for imaging, together with a pharmaceutically acceptable radiological vehicle. The radiological vehicle should be suitable for injection or aspiration, such as human serum albumin; aqueous buffer solutions, phosphate, citrate, bicarbonate, etc; sterile water physiological saline; and balanced ionic solutions containing chloride and or dicarbonate salts or normal blood plasma cautions such as calcium potassium, sodium and magnesium. The concentration of the imaging agent or the therapeutic agent in the radiological vehicle should be sufficient to provide satisfactory imaging. Appropriate dosages have been described e.g. in WO 2020/165420. The term "sample" refers to a sample of a bodily fluid, preferably a sample of blood, plasma, serum, saliva, sputum, urine, or a sample comprising separated cells or to a sample from a tissue or an organ. More preferably, the sample is a cancer sample, in particular a tumor or metastasis sample, which may be obtained e.g. by biopsy. Separated cells may be obtained from the body fluids, such as lymph, blood, plasma, serum, liquor and other, or from the tissues or organs by separating techniques such as centrifugation or cell sorting. More preferably, the sample is a sample known or suspected to comprise cancer cells. The sample can be obtained from the patient by any method deemed appropriate by the skilled person, preferably by routine techniques which are well known to the person skilled in the art, e.g., venous or arterial puncture or open biopsy including aspiration of tissue or cellular material from a subject. For those areas which cannot be easily reached via an open biopsy, a surgery and, preferably, minimal invasive surgery can be performed.
Further details regarding the synthetic procedures to prepare the compounds of the present invention are described in the example section
Summarizing the findings of the present invention, the following embodiments are particularly preferred:
1. A compound of formula (1)
Figure imgf000049_0001
or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is H or -CH3, preferably H, wherein R2, R3 and R4 are independently of each other, selected from the group consisting of -CO2H, -SO2H, -SO3H, -OSO3H, -PO2H2, -PO3H2, -OPO3H2, - CONH2 and -C0NH0H, wherein L is a suitable linker, linking A and NH, preferably L is a linking group selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q-C(=O)-, -(Q3)q-C(=S)-, -(Q3)q- NH-C(=S)-, wherein Q3 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is 0 or 1, and wherein n is 0 or 1, wherein XAA1 is an amino acid moiety having the structure (A)
Figure imgf000050_0001
with Q1 being a residue comprising an optionally substituted styryl moiety, said residue preferably having the structure -(CHR5)m-CH=CH-Ph, where m equals 0, 1, 2, or 3, each R5, independently of others, is selected from the group consisting of H, alkyl and aryl, and Ph is phenyl optionally substituted with one to five substituents selected from alkyl and aryl groups,
Q2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
A is a chelator residue derived from a chelator selected from the group consisting of
1.4.7.10-tetraazacyclododecane-N,N',N",N "'-tetraacetic acid (=DOTA), N,N"-bis[2- hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid, 1,4,7- triazacyclononane-l,4,7-triacetic acid (=NOTA), 2-(4,7-bis(carboxymethyl)- 1,4,7- triazonan-l-yl)pentanedioic acid (=N0DAGA), 2-(4,7,10-tris(carboxymethyl)-
1.4.7.10-tetraazacyclododecan-l-yl)pentanedioic acid (=DOTAGA), 1,4,7- triazacyclononane phosphinic acid (=TRAP), l,4,7-triazacyclononane-l-[methyl(2- carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (=NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene-3,6,9-triacetic acid (=PCTA), l,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- m ethylperhydro- 1 ,4-diazepine (=AAZTA), 1 ,4,7, 10-tetra-azacyclododecan- 1,4,7,10- tetra-azidoethylacetic acid (=DOTAZA), Nl-(5-aminopentyl)-Nl-hydroxy-N4-(5-(N- hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4- oxobutanamido)pentyl)succinamide (=DFO), Nl-[5-(Acetylhydroxyamino)pentyl]- N26-(5-aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23 -tetraoxo-5,11, 16,22- tetraazahexacosanediamide (=DFO*), oxo-DFO*, N,N'-bis[(6-carboxy-2- pyridil)methyl]-4,13-diaza-18-crown-6 (macropa), 4-Amino-6-((16-((6- carboxypyridin-2-yl)methyl)-l,4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7- yl)methyl)picolinic acid (=Macropa-NH2), 2,2'-(ethane-l,2-diylbis(((8- hydroxyquinolin-2-yl)methyl)azanediyl)) diacetic acid (=H4octox), 6,6'-((9-hydroxy- l,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7- diyl)bis(methylene))dipicolinic acid (=H2bispa), dimethyl 9-hydroxy-3,7-bis((8- hydroxyquinolin-2-yl)methyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-l,5- dicarboxylate (=H2bispox), 6,6'-(((pyridine-2,6-diylbis(methylene))- bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid (=H4py4pa), Diethylenetriaminepentaacetic acid (=DTPA), Trans-cyclohexyl- diethylenetriaminepentaacetic acid (=CHX-DTPA), 1,4,7, 10-tetraazacyclododecane- 1,4,7-triacetic acid (=DO3A), l-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (=oxo-Do3 A), 1,4,7, 10-Tetrakis(carbamoylmethyl)- 1,4,7, 10-tetraazacyclododecane
(=D0TAM), N,N'-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N'-diacetic acid (=H4Octapa), 1,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (=HEHA), , l,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N'm-pentaacetic acid (=PEPA), cyclam-based ligands with N-methyl, N-(4-aminobenzyl), N-phosphinate- and phosphonate-based substituents, wherein oxo-DFO* has the structure:
Figure imgf000051_0001
The compound of embodiment 1, wherein the compound has the structure (la),
Figure imgf000051_0002
preferably, the structure (laa)
Figure imgf000051_0003
wherein R5, R6, R7, R8, R9, and R10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl. 3. The compound of embodiment 1 or 2, wherein the compound has the structure (lb),
Figure imgf000052_0001
preferably, the structure (Ibb)
Figure imgf000052_0002
4. The compound of any one of embodiments 1 to 3, wherein A is a chelator residue having a structure selected from the group consisting of
Figure imgf000052_0003
Figure imgf000053_0001
The compound of any of one of embodiments 1 to 4, wherein R3, R2 and R4 are -CO2H and R1 is H. The compound of any one of embodiments 1 to 5, wherein Q2 is
Figure imgf000053_0002
preferably
Figure imgf000054_0001
wherein R11, R12, R13 and R14 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl. The compound of embodiment 6, wherein Q2 is
Figure imgf000054_0002
preferably
Figure imgf000054_0003
The compound of any one of embodiments 1 to 7 having the structure
Figure imgf000054_0004
or the structure
Figure imgf000055_0001
Figure imgf000055_0002
10. The compound of any one of embodiments 1 to 7 having the structure
Figure imgf000056_0001
5 or
Figure imgf000057_0001
11 . Complex comprising
(a) a radionuclide, and (b) the compound of any one of embodiments 1 to 10 or a pharmaceutically acceptable salt or solvate thereof.
12. The complex of embodiment 11, wherein, the radionuclide is selected from the group consisting 18F, 89Zr, 43Sc, 44Sc, 47Sc, 45Ti, 55Co, mIn, 86Y, 90Y, 66Ga, 67Ga, 68Ga, 177Lu, "mTc, 60Cu, 61Cu, 62Cu, 64Cu, 66Cu, 67Cu, 149Tb, 152Tb, 155Tb, 153Sm, 161Tb, 166Ho, 153Gd,
155Gd, 157Gd, 211At, 213Bi, 225 Ac, 230U, 223Ra, 224Ra, 165Er, 52Fe, 59Fe, 186Re, 188Re, 227Th, 133La, 72 As and radionuclides of Pb, such as 203Pb and 212Pb. 13. Method for preparing a compound of formula (1)
Figure imgf000057_0002
or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is H or -CH3, preferably H, wherein R2, R3 and R4 are CO2H,. wherein XAA1 is an amino acid moiety having the structure (A)
Figure imgf000058_0001
with Q1 being a residue comprising an optionally substituted styryl moiety, said residue preferably having the structure -(CHR5)m-CH=CH-Ph, where m equals 0, 1, 2, 3, each R5, independently of others, is selected from the group consisting of H, alkyl and aryl, and Ph is phenyl optionally substituted with one to five substituents selected from alkyl and aryl groups, wherein L is a suitable linker, linking A and NH, preferably L is a linking group selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q-C(=O)-, -(Q3)q-C(=S)-, -(Q3)q- NH-C(=S)-, wherein Q3 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is 0 or 1, and wherein n is 0 or 1,
Q2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
A is a chelator residue derived from a chelator selected from the group consisting of
1.4.7.10-tetraazacyclododecane-N,N',N",N "'-tetraacetic acid (=DOTA), N,N"-bis[2- hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid, 1,4,7- triazacyclononane-l,4,7-triacetic acid (=NOTA), 2-(4,7-bis(carboxymethyl)- 1,4,7- triazonan-l-yl)pentanedioic acid (=N0DAGA), 2-(4,7,10-tris(carboxymethyl)-
1.4.7.10-tetraazacyclododecan-l-yl)pentanedioic acid (=DOTAGA), 1,4,7- triazacyclononane phosphinic acid (=TRAP), l,4,7-triazacyclononane-l-[methyl(2- carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (=NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene-3,6,9-triacetic acid (=PCTA), l,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- m ethylperhydro- 1 ,4-diazepine (=AAZTA), 1 ,4,7, 10-tetra-azacyclododecan- 1,4,7,10- tetra-azidoethylacetic acid (=DOTAZA), Nl-(5-aminopentyl)-Nl-hydroxy-N4-(5-(N- hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4- oxobutanamido)pentyl)succinamide (=DFO), Nl-[5-(Acetylhydroxyamino)pentyl]- N26-(5-aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23 -tetraoxo-5,11, 16,22- tetraazahexacosanediamide (=DFO*), oxo-DFO*, N,N'-bis[(6-carboxy-2- pyridil)methyl]-4,13-diaza-18-crown-6 (macropa), 4-Amino-6-((16-((6- carboxypyridin-2-yl)methyl)-l,4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7- yl)methyl)picolinic acid (=Macropa-NH2), , 2,2'-(ethane-l,2-diylbis(((8- hydroxyquinolin-2-yl)methyl)azanediyl)) diacetic acid (=H4octox), 6,6'-((9-hydroxy- l,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7- diyl)bis(methylene))dipicolinic acid (=H2bispa), dimethyl 9-hydroxy-3,7-bis((8- hydroxyquinolin-2-yl)methyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-l,5- dicarboxylate (=H2bispox), 6,6'-(((pyridine-2,6-diylbis(methylene))- bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid (=H4py4pa), Diethylenetriaminepentaacetic acid (=DTPA), Trans-cyclohexyl- diethylenetriaminepentaacetic acid (=CHX-DTPA), 1,4,7, 10-tetraazacyclododecane- 1,4,7-triacetic acid (=D03A), l-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (=oxo-Do3 A), 1,4,7, 10-Tetrakis(carbamoylmethyl)- 1,4,7, 10-tetraazacyclododecane (=DOTAM), N,N'-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N'-diacetic acid (=H4Octapa), 1,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (=HEHA), 1,4,7,10,13 -pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (=PEPA), cyclam-based ligands with N-methyl, N-(4-aminobenzyl), N-phosphinate- and phosphonate-based substituents, wherein oxo-DFO* has the structure:
Figure imgf000059_0001
the method comprising:
(aa) attaching Fmoc-Lys(NPG' )-0H to a suitable resin, preferably a 2-chloro- trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG1 is a suitable protecting group, preferably being orthogonal to Fmoc and 2-CT linker of the resin, more preferably Alloc,
(bb) reacting H-G1U(OPG2)-OPG2 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming the activated Glu compound of formula (II)
Figure imgf000059_0002
wherein PG2 is a suitable protecting group, preferably being orthogonal to Fmoc and Alloc, more preferably tBu
(cc) reacting the compound of formula (II) with the Lys modified resin according to step (aa) and removing PG1, thereby forming the modified resin
Figure imgf000060_0001
(dd) reacting the modified resin according to (cc) with Fmoc-(XAA1)-OH, and removing the Fmoc group thereby forming the modified resin
Figure imgf000060_0002
(ee) reacting the modified resin according
Fmoc — NH— CH
Figure imgf000060_0003
and removing the Fmoc group,
(ff) linking the free amino group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L,
(gg) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1). Method according to embodiment 13, wherein step (aa) comprises
(aal) attaching attaching Fmoc-Lys(NPG1)-OH to the resin, preferably the 2-chloro- trityl resin in the presence of A,A-ethyldiisopropylamine (DIPEA), and
(aa2) removing the Fmoc group in the presence of piperidine in DMF. 15. Method according to embodiment 13 or 14, wherein step (bb) is carried out in the presence of a base, preferably DIPEA.
16. Method according to any one of embodiments 13 to 15, wherein the PG1 is an Alloc protecting group and the group is removed by reaction with TPP palladium in the presence of morpholine.
17. Method according to any one of embodiments 13 to 16, wherein step (ee) comprises
(eel) reacting the modified resin according to (dd) with Fmoc-
Figure imgf000061_0001
(ee2) removing the Fmoc group. wherein (eel) is carried out in the presence of a suitable activation reagent, preferably HBTU or Oxyma Pure and DIC.
18. Method according to any one of embodiments 13 to 17, wherein in step (ff) A-(L)n is provided in the form of an NHS ester and is subsequently attached to free amino group, thereby, preferably, the group A-(L)n-NH- is formed which is more preferably A-(Q3)q-C(=O)-NH-, or
A-(L)n is provided in the form of an activated isothiocyanate with which the free amino group is then reacted, thereby, preferably, the group A-(L)n-NH-, more preferably A-(Q3)q-NH-C(=S)-NH- is formed.
19. Method according to any one of embodiments 13 to 18, wherein step (gg) is carried out in the presence of trifluoroacidic acid. 0. Method for preparing a compound of formula (1)
Figure imgf000061_0002
or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is H or -CH3, preferably H, wherein R2, R3 and R4 are independently of each other, selected from the group consisting of -CO2H, -SO2H, -SO3H, -OSO3H, -PO2H2, -PO3H2, -OPO3H2, - CONH2 and -C0NH0H, wherein L is a suitable linker, linking A and NH, preferably L is a linking group selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q-C(=O)-, -(Q3)q-C(=S)-, -(Q3)q- NH-C(=S)-, wherein Q3 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is 0 or 1, and wherein n is 0 or 1, wherein XAA1 is an amino acid moiety having the structure (A)
Figure imgf000062_0001
wherein Q1 is a residue comprising an optionally substituted styryl moiety, said residue preferably having the structure -(CHR5)m-CH=CH-Ph, where m equals 0, 1, 2, or 3, each R5, independently of others, is selected from the group consisting of H, alkyl and aryl, and Ph is phenyl optionally substituted with one to five substituents selected from alkyl and aryl groups,
Q2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
A is a chelator residue derived from a chelator selected from the group consisting of
1.4.7.10-tetraazacyclododecane-N,N',N",N "'-tetraacetic acid (=DOTA), N,N"-bis[2- hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid, 1,4,7- triazacyclononane-l,4,7-triacetic acid (=NOTA), 2-(4,7-bis(carboxymethyl)- 1,4,7- triazonan-l-yl)pentanedioic acid (=N0DAGA), 2-(4,7,10-tris(carboxymethyl)-
1.4.7.10-tetraazacyclododecan-l-yl)pentanedioic acid (=DOTAGA), 1,4,7- triazacyclononane phosphinic acid (=TRAP), l,4,7-triazacyclononane-l-[methyl(2- carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (=NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene-3,6,9-triacetic acid (=PCTA), l,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- m ethylperhydro- 1 ,4-diazepine (=AAZTA), 1 ,4,7, 10-tetra-azacyclododecan- 1,4,7,10- tetra-azidoethylacetic acid (=DOTAZA), Nl-(5-aminopentyl)-Nl-hydroxy-N4-(5-(N- hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4- oxobutanamido)pentyl)succinamide (=DFO), Nl-[5-(Acetylhydroxyamino)pentyl]- N26-(5-aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23 -tetraoxo-5,11, 16,22- tetraazahexacosanediamide (=DFO*), oxo-DFO*, N,N'-bis[(6-carboxy-2- pyridil)methyl]-4,13-diaza-18-crown-6 (macropa), 4-Amino-6-((16-((6- carboxypyridin-2-yl)methyl)-l,4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7- yl)methyl)picolinic acid (=Macropa-NH2), 2,2'-(ethane-l,2-diylbis(((8- hydroxyquinolin-2-yl)methyl)azanediyl)) diacetic acid (=H4octox), 6,6'-((9-hydroxy- l,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7- diyl)bis(methylene))dipicolinic acid (=H2bispa), dimethyl 9-hydroxy-3,7-bis((8- hydroxyquinolin-2-yl)methyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-l,5- dicarboxylate (=H2bispox), 6,6'-(((pyridine-2,6-diylbis(methylene))- bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid (=H4py4pa), Diethylenetriaminepentaacetic acid (=DTPA), Trans-cyclohexyl- diethylenetriaminepentaacetic acid (=CHX-DTPA), 1,4,7, 10-tetraazacyclododecane- 1,4,7-triacetic acid (=D03A), l-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (=oxo-Do3 A), 1,4,7, 10-Tetrakis(carbamoylmethyl)- 1,4,7, 10-tetraazacyclododecane
(=DOTAM), N,N'-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N'-diacetic acid (=H4Octapa), 1,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (=HEHA)), 1 ,4,7, 10, 13-pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (=PEPA), cyclam-based ligands with N-methyl, N-(4-aminobenzyl), N-phosphinate- and phosphonate-based substituents, wherein oxo-DFO* has the structure:
Figure imgf000063_0001
the method comprising:
(a) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(b) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000063_0002
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu, (c) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin
Figure imgf000064_0001
preferably
Figure imgf000064_0002
more preferably
Figure imgf000064_0003
more preferably
Figure imgf000064_0004
(d) reacting the modified resin according to (c) with Fmoc-(XAAi)-OH, and removing the Fmoc group thereby forming the modified resin
Figure imgf000065_0001
preferably
Figure imgf000065_0002
more preferably
Figure imgf000065_0003
more preferably H
Xaa 1
Figure imgf000066_0002
and removing the Fmoc group,
(f) linking the free amino group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L,
(g) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1). Method according to embodiment 20, wherein step (a) comprises
(al) attaching attaching Fmoc-Glu(OPG2)-OH to the resin, preferably the 2-chloro- trityl resin in the presence of A,A-ethyldiisopropylamine (DIPEA), and
(a2) removing the Fmoc group in the presence of piperidine in DMF. Method according to embodiment 20 or 21, wherein step (b) is carried out in the presence of a base, preferably DIPEA. Method according to any one of embodiments 20 to 22, wherein no Alloc protecting group is used. Method according to any one of embodiments 20 to 23, wherein step (e) comprises
(el) reacting the modified resin according to (d) with
Figure imgf000066_0001
(e2) removing the Fmoc group, wherein (el) is carried out in the presence of a suitable activation reagent, preferably HBTU or Oxyma Pure and DIC. Method according to any one of embodiments 20 to 24, wherein in (f) A-(L)n is provided in the form of an NHS ester and is subsequently attached to free amino group, thereby, preferably, the group A-(L)n-NH- is formed which is more preferably A-(Q3)q-C(=O)-NH-, or
A-(L)n is provided in the form of an activated isothiocyanate with which the free amino group is then reacted, thereby, preferably, the group A-(L)n-NH-, more preferably A-(Q3)q-NH-C(=S)-NH- is formed. Method according to any one of embodiments 20 to 25, wherein in step (g) is carried out in the presence of trifluoroacetic acid. Method according to any one of embodiments 20 to 26, wherein step (c) comprises
(cl) reacting the compound of formula (Ila) with the Glu modified resin according to step (a), and
(c2) removing the Fmoc group, wherein (cl) is preferably carried out in the presence of DIPEA. Compound of formula (1), obtained or obtainable by the method according to any one of embodiments 13 to 27. Method for preparing a resin bound PSMA binding moiety having the structure
Figure imgf000067_0001
preferably
Figure imgf000067_0002
more preferably
Figure imgf000068_0001
more preferably
Figure imgf000068_0002
as intermediate product for the solid phase synthesis of PSMA ligands, preferably for the synthesis of a PSMA ligand according to embodiment 1 to 11, the method comprising the steps,
(d) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-Chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(e) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000068_0003
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu,
(f) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin. pharmaceutical composition comprising a compound of any one of embodiment 1 to0 or a complex of embodiment 11 or 12. 31. A compound of any one of embodiment 1 to 10 or a complex of embodiment 11 or 12 for use in medicine.
32. A compound of any one of embodiment 1 to 10 or a complex of embodiment 11 or 12 for use in treating and/or preventing PSMA expressing cancer, in particular prostate cancer and/or metastases thereof.
33. Compound of any one of embodiment 1 to 10 or a complex of embodiment 11 or 12 or a pharmaceutical composition of embodiment 30 for use in diagnostics.
34. A compound of any one of embodiment 1 to 10 or a complex of embodiment 11 or 12 for use in the diagnosis of cancer, preferably of PSMA expressing cancer, in particular of prostate cancer and/or metastases thereof.
35. The compound or complex of embodiment 33, wherein said diagnosis is practised on the human or animal body.
36. A compound of any one of embodiments 1 to 10 or a complex of embodiment 11 or 12 for use in diagnosing a PSMA expressing cancer in a patient, said diagnosing comprising
(i) determining binding of said compound or complex to a tissue of said subject;
(ii) comparing the binding determined in step (i) to a reference;
(iii) identifying a difference in step (ii); and
(iv) diagnosing said cancer based on the result of step (iii).
35. A method for diagnosing a PSMA expressing cancer in a sample of a patient, the method comprising
(I) determining binding of a compound of any one of embodiments 1 to 10 or a complex of embodiment 11 or 12 to said sample; and
(II) diagnosing said cancer based on the result of step (I).
36. The method of embodiment 35, further comprising step (la) comparing the binding determined in step (I) to a reference; wherein step (II) comprises diagnosing said cancer based on the result of step (la).
37. A method for treating a PSMA expressing cancer in a patient, the method comprising
(A) contacting said patient with a compound of any one of embodiments 1 to 10 or a complex of embodiment 11 or 12 to said sample; and
(B) thereby treating said cancer in said patient.
38. Use of a compound of any one of embodiments 1 to 10 or a complex of embodiment 11 or 12 for the manufacture of a diagnostic for diagnosing a PSMA expressing cancer and/or of a medicament for treating a PSMA expressing cancer. Fig. 1: Synthetic scheme for the conjugation of Fmoc-TXA for Pl 7.
Fig. 2: Synthetic scheme for the conjugation of the DOTA chelator to obtain P17/P18.
Fig. 3: Synthesis Scheme for the Synthesis ofP17 via method 1.
Fig. 4: Synthesis Scheme for the Synthesis of P18 via method 1.
Fig. 5: Synthesis of urea intermediate via method 2.
Fig. 6: Synthesis Scheme for the Synthesis of P17 via method 2.
Fig. 7: Synthesis Scheme for the Synthesis of P18 via method 2.
Fig. 8: Chemical structures of (a) P17 and (b) P17-Macropa.
Fig. 9: Chemical structures of (a) Pl 8, (b) P18-Macropa, and (c) P18-HEHA.
Fig. 10: Chemical structure of PSMA-617.
Fig. 11: Structural characterization of PSMA complexes. (A) The Fo-Fc omit map (gray) is contoured at 3.0 c and inhibitors are shown in stick representation. The active-site zinc ions are shown as black spheres. Notice the absent electron density for the DOTA chelator of 617 implying its positional flexibility due to missing interactions with PSMA. (B) Superposition of inhibitors in PSMA/inhibitor complexes. PSMA molecules in individual complexes were superpositioned on corresponding Ca atoms. While positioning of P17/P18 is virtually identical, there are marked differences in positioning of linker and chelator moieties of P17 (gray) and 617 (black). (C, D) Flexibility of the entrance lid of PSMA in PSMA/inhibitor complexes. Inhibitors are in stick representation, PSMA is shown in surface representation (light gray) with residues forming the entrance lid (Thr538 - Gly548) colored dark gray and Trp541 colored black. For the PSMA/617 complex (C) the lid is in in partially closed conformation restricting the diameter of the entrance funnel to app 12 A, while the open lid conformation in the PSMA/P17 complex (D) increases in the entrance funnel diameter to app 20 A. (E) Flexibility of the entrance lid is required to accommodate inhibitors with different distal moieties. PSMA/P17 and PSMA/617 complexes were superpositioned on corresponding Ca atoms. Putative binding of P17 (stick representation) in PSMA enzyme derived from the PSMA/617 (surface representation) is shown. Notice the steric clash between the inhibitor and the enzyme (inhibitor is in part “buried” inside surface contours of the entrance lid) pointing towards the necessity of the lid repositioning for P17 binding. (F) Flexibility of the entrance lid is required to accommodate inhibitors with different distal moieties. PSMA/P17 and PSMA/617 complexes were superpositioned on corresponding Ca atoms. P17 is shown in stick representation, PSMA in surface representation and the entrance lid as cartoon colored light gray and black for PSMA/P17 and PSMA/617, respectively. Notice differences in the lid conformation. Repositioning by up to 6 A is required to accommodate Pl 7. (G) DOTA function of Pl 7 (stick representation) engages the side chain of Trp541 of the entrance lid (cartoon representation) via an array of CH/n bonds (broken lines). PSMA is shown in surface representation. PSMA inhibitor complexes were deposited at the Protein databank with accession codes 8B0W, 8BO8, and 8BOL for 617, Pl 7, and Pl 8 respectively. Distances are shown in Angstroms.
Fig. 12. Graphs representing the uptake of radiolabeled PSMA compounds in PC3-PIP and PC3-flu tumors and various other tissues and organs at 1 h and 2 h after injection. (A) [177Lu]Lu- P17; (B) [177LU]LU-P18; (C) [177LU]LU-PSMA-617. The data are reported as average ± SD [% lA/g] values from each group of mice (n=3).
EXAMPLES:
General Synthesis Outline P17/P18
The solid-phase synthesis of the peptidomimetic glutamate-urea-lysine binding motif is summarized in Scheme 1A, IB, 1C and ID. The ensuing coupling of the linker was performed according to standard fluorenylmethoxycarbonyl (Fmoc) protocol (Scheme 2A and 2B for P17 as well as Scheme 2AA for Pl 8, respectively). Finally, conjugation of the DOTA chelator (Scheme 3A and 3B) was realized using HBTU-activated DOTA-//7.s(/Bu)ester or an active DOTA-NHS ester.
Binding Motif Synthesis P17/P18
Scheme 1A: Synthesis of the peptidomimetic glutamate-urea-lysine binding motif via analogical strategy as the one for PSMA-617
Figure imgf000071_0001
2-Chloro-trityl resin (2-CT resin, 0.3 mmol, substitution capacity 1.22 mmol/g, 100-200 MESH) was first agitated in dry dichloromethane (DCM) for 45 min and then washed with dry DCM followed by reaction with 1.2 equiv Alloc- (As-allyloxycarbonyl) and Fmoc (Na- fhiorenylmethoxycarbonyl)-protected L-lysine [Fmoc-L-Lys(Alloc)-OH, 1], and 4.8 equiv A,A-ethyldiisopropylamine (DIPEA) in 3 mL of dry DCM. The coupling of the first protected amino acid on the resin proceeded over the course of 16 h with gentle agitation.
The lysine-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, methanol (MeOH), and DIPEA in a ratio of 17:2: 1, respectively. Selective removal of the Fmoc-protecting group was realized by washing with a mixture of A,A-dimethylformamide (DMF) and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to get product 3. In the next step, 10 equiv bis(/Bu)-L-glutamate hydrochloride [H-Glu(O/Bu)-O/Bu HCl, 3.0 mmol] were used for generation of the isocyanate of the glutamyl moiety. An appropriate amount of /Bu-protected glutamate was dissolved in 200 mL of dry DCM followed by, shortly afterward, the addition of 3 mL of DIPEA. This solution was added dropwise over 4 h to a flask with 1.0 mmol of ice-cooled bis(trichloromethyl)carbonate (BTC, 0 °C) in 5 mL of dry DCM. The lysine-immobilized resin 3 was added afterward in one portion to the solution of the isocyanate of the glutamyl moiety and stirred for 16 h.
The product 4 was filtered off and washed with DCM. The cleavage of the Alloc-protecting group was realized by reaction with 0.3 equiv tetrakis(triphenyl)palladium (TPP palladium) in the presence of 30 equiv morpholine in dry DCM for 2 h. The reaction was performed in the dark using aluminum foil. To remove residuals of the palladium catalysator, the resin was washed with 1% DIPEA in DMF and subsequently with a solution of sodium diethyldithiocarbamate trihydrate (15 mg/mL) in DMF (10 times for 5 min). The resin- immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
Scheme IB: Synthesis of the peptidomimetic glutamate-urea-lysine binding motif via adapted strategy as the one for PSMA-617
Figure imgf000072_0001
5 g 2-CT resin (6.1 mmol, substitution capacity 1.22 mmol/g, 100-200 MESH) were first agitated in dry DCM for 45 min and then washed with dry DCM followed by reaction with 1.2 equiv Fmoc-L-Lys(Alloc)-OH (1), and 4.8 equiv DIPEA in 150 mL of dry DCM. The coupling of the first protected amino acid on the resin proceeded over the course of 16 h with gentle agitation.
The lysine-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, MeOH, and DIPEA in a ratio of 17:2: 1, respectively. Selective removal of the Fmoc-protecting group was realized by washing three times with 30 mL of a mixture of DMF and piperidine in a ratio of 1 : 1 in order to get product 3.
In the next step, 2.8 mmol BTC were dissolved in 3 mL of dry DCM and combined with 8.5 mmol H-Glu(OtBu)-OtBu HC1 dissolved in 15 mL dry DCM. Shortly afterward, the solution of 12 mL DIPEA in 10 mL DCM was added dropwise over 1 h to a flask with ice/acetone- cooled BTC/glutamate (-10 °C). The 2 g of the lysine-immobilized resin 3 was added afterward in one portion to the solution of the isocyanate of the glutamyl moiety and stirred for 16 h.
The product 4 was filtered off and washed with DCM. The cleavage of the Alloc-protecting group was realized by reaction with 0.3 equiv TPP palladium in the presence of 30 equiv morpholine in dry DCM for 2 h. The reaction was performed in the dark using aluminum foil. To remove residuals of the palladium catalysator, the resin was washed with 1% DIPEA in DMF and subsequently with a solution of sodium diethyldithiocarbamate trihydrate (15 mg/mL) in DMF. The resin-immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
Scheme 1C: Synthesis of the peptidomimetic glutamate-urea-lysine binding motif via adapted new strategy
Figure imgf000073_0001
5 g 2-CT resin (6.1 mmol, substitution capacity 1.22 mmol/g, 100-200 MESH) were first agitated in dry DCM for 45 min and then washed with dry DCM followed by reaction with 1.2 equiv Fmoc-L-Lys(Alloc)-OH (1), and 4.8 equiv DIPEA in 150 mL of dry DCM. The coupling of the first protected amino acid on the resin proceeded over the course of 16 h with gentle agitation.
The lysine-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, MeOH, and DIPEA in a ratio of 17:2:1, respectively. Selective removal of the Fmoc-protecting group was realized by washing three times with 30 mL of a mixture of DMF and piperidine in a ratio of 1 : 1 in order to get product 3.
In the next step, 27.0 mmol H-Glu(OtBu)-OtBu HC1 was combined with 27.0 mmol 1,1'- carbonyldiimidazol (CDI) and 27.0 mmol DIPEA. This mixture was diluted with 50 mL of dry DMF and stirred at RT for 1 h. The lysine-immobilized resin 3 was added afterward in one portion to the solution of the CDLadduct of the glutamyl moiety and stirred for 16 h.
The product 4 was filtered off and washed with DCM. The cleavage of the Alloc-protecting group was realized by reaction with 0.3 equiv TPP palladium in the presence of 30 equiv morpholine in dry DCM for 2 h. The reaction was performed in the dark using aluminum foil. To remove residuals of the palladium catalysator, the resin was washed with 1% DIPEA in DMF and subsequently with a solution of sodium diethyldithiocarbamate trihydrate (15 mg/mL) in DMF. The resin-immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
Scheme ID: Synthesis of the peptidomimetic glutamate-urea-lysine binding motif via adapted new strategy
Figure imgf000074_0001
5 g 2-CT resin (6.1 mmol, substitution capacity 1.22 mmol/g, 100-200 MESH) was first agitated in dry DCM for 45 min and then washed with dry DCM followed by reaction with 1.2 equiv Fmoc-Glu(OtBu)-OH (1), and 4.8 equiv DIPEA in 150 mL of dry DCM. The coupling of the first protected amino acid on the resin proceeded over the course of 16 h with gentle agitation.
The glutamate-immobilized resin 2 was then washed with DCM with unreacted chlorotrityl groups remaining in the resin blocked with a mixture of DCM, MeOH, and DIPEA in a ratio of 17:2: 1, respectively. Selective removal of the Fmoc-protecting group was realized by washing three times with 30 mL of a mixture of DMF and piperidine in a ratio of 1 : 1 in order to get product 3.
In the next step, 16.3 mmol H-Lys(Fmoc)-OtBu was combined with 16.3 mmol 1,1'- carbonyldiimidazol (CDI) and 16.6 mmol DIPEA. This mixture was diluted with 100 mL of dry DMF and stirred at RT for 1 h. The glutamate-immobilized resin 3 was added afterward in one portion to the solution of the CDI-adduct of the lysine moiety and stirred for 16 h.
The product 4 was filtered off and washed with DCM and DMF. Selective removal of the Fmoc- protecting group was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min. The resin-immobilized and /Bu-protected binding motif 5 was dried under vacuum and split into three portions.
Linker Coupling P17
Scheme 2A: Coupling of linker binding blocks via analogical strategy as the one for PSMA- 617
Figure imgf000075_0001
Relative to the resin (0.1 mmol), 4 equiv Fmoc-3-styryl-L-Ala (0.400 mmol) were activated with 3.92 equiv HBTU (O-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 0.392 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 5 and agitated at RT for 1 h. Selective removal of the Fmoc- protecting group from the product 6 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 7.
In the next step, 4 equiv Fmoc-TXA (0.400 mmol) were reacted with 3.92 equiv HBTU (0.392 mmol) and 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 7 and agitated at RT for 1 h. Selective removal of the Fmoc-protecting group from the product 8 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 9.
Scheme 2B: Coupling of linker binding blocks via adapted new strategy
Figure imgf000076_0001
Relative to the resin (0.1 mmol), 4 equiv Fmoc-3-styryl-L-Ala (0.400 mmol) were activated with 3.92 equiv HBTU (0.392 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 5 and agitated at RT for 1 h. Selective removal of the Fmoc- protecting group from the product 6 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 7.
In the next step, 5 equiv Fmoc-TXA (0.500 mmol) were reacted with 5 equiv Oxyma Pure (0.500 mmol) and 5 equiv DIC (l,l'-carbonyldiimidazol, 0.500 mmol) in 10 mL dry DMF:DCM in a ratio 1 : 1. The solution was added to the DMF pre-swollen immobilized pharmacophore 7 and agitated at RT for 1 h. Selective removal of the Fmoc-protecting group from the product 8 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 9.
Scheme 2C: Coupling of linker binding blocks via adapted new strategy
Figure imgf000077_0001
Relative to the resin (0.1 mmol), 4 equiv Fmoc-3-styryl-L-Ala (0.400 mmol) were activated with 3.92 equiv HBTU (0.392 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 5 and agitated at RT for 1 h. Selective removal of the Fmoc- protecting group from the product 6 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 7.
In the next step, 5 equiv Fmoc-TXA (0.500 mmol) were reacted with 5 equiv Oxyma Pure (0.500 mmol) and 5 equiv DIC (l,l'-carbonyldiimidazol, 0.500 mmol) in 10 mL dry DMF:DCM in a ratio 1 : 1. The solution was added to the DMF pre-swollen immobilized pharmacophore 7 and agitated at RT for 1 h. Selective removal of the Fmoc-protecting group from the product 8 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 9.
Linker Coupling P18
Scheme 2AA: Coupling of linker binding blocks via analogical strategy as the one for PSMA- 617
Figure imgf000078_0001
Relative to the resin (0.1 mmol), 4 equiv Fmoc-3-styryl-L-Ala (0.400 mmol) were activated with 3.92 equiv HBTU (O-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 0.392 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 5 and agitated at RT for 1 h. Selective removal of the Fmoc- protecting group from the product 6 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 7.
In the next step, 4 equiv Fmoc-AMBA (4-(Fmoc-aminomethyl)benzoic acid, 0.400 mmol) were reacted with 3.92 equiv HBTU (0.392 mmol) and 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 7 and agitated at RT for 1 h. Selective removal of the Fmoc- protecting group from the product 8 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 9.
Linker Coupling P18
Scheme 2AAA: Coupling of linker binding blocks via analogical strategy as the one for PSMA-
617
Figure imgf000079_0001
Relative to the resin (0.1 mmol), 4 equiv Fmoc-3-styryl-L-Ala (0.400 mmol) were activated with 3.92 equiv HBTU (O-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 0.392 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 5 and agitated at RT for 1 h. Selective removal of the Fmoc- protecting group from the product 6 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 7.
In the next step, 4 equiv Fmoc-AMBA (4-(Fmoc-aminomethyl)benzoic acid, 0.400 mmol) were reacted with 3.92 equiv HBTU (0.392 mmol) and 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized pharmacophore 7 and agitated at RT for 1 h. Selective removal of the Fmoc- protecting group from the product 8 was realized by washing with a mixture of DMF and piperidine in a ratio of 1 : 1 once for 2 min and then once again for 5 min in order to obtain the product 9.
Chelator Conjugation P17/P18
Scheme 3A: Conjugation of DOTA-//7.s(/Bu)ester via analogical strategy as the one for PSMA- 617
Figure imgf000080_0001
Relative to the resin (0.1 mmol), 4 equiv DOTA-//7.s(/Bu)ester (2-(4,7,10-tris(2-(t-butoxy)-2- oxoethyl)-l,4,7,10-tetraazacyclododecan-l-yl)acetic acid, 0.400 mmol) were activated with 3.95 equiv HBTU (0.395 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry
DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized compound 9 and agitated at RT for 3 h. The final products, i.e. P17/P18, were obtained by agitation and subsequent cleavage from the resin and /Bu deprotection within 3 h with a mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water in a ratio of 95:2.5:2.5, respectively. The mixture of solvents from the product was evaporated and the crude compound was dissolved in acetonitrile (ACN) and water in a ratio of 1 : 1 for subsequent reversed-phase high-performance liquid chromatography (RP-HPLC) purification. Scheme 3B: Conjugation of DOTA-//7.s(/Bu)-NHS ester via analogical strategy as the one for PSMA-617
Figure imgf000081_0001
Relative to the resin (0.1 mmol), 1.5 equiv DOTA-//7.s(/Bu)NHS ester (0.150 mmol) was dissolved in 3 mL dry DMF and 1.5 equiv TEA (A,A,A-trimethylamine, 0.150 mmol). Two min after the addition of TEA, the solution was added to the DMF pre-swollen immobilized compound 9 and agitated at RT for 16 h. The final product, i.e. P17/P18, was obtained by agitation and subsequent cleavage from the resin and /Bu deprotection within 3 h with a mixture consisting of TFA, TIPS, and water in a ratio of 95:2.5:2.5, respectively. The mixture of solvents from the product was evaporated and the crude compound was dissolved in ACN and water in a ratio of 1 : 1 for subsequent reversed- phase high-performance liquid chromatography (RP-HPLC) purification.
Scheme 3C: Conjugation of DOTA-//7.s(/Bu)ester via analogical strategy as the one for PSMA- 617
Figure imgf000082_0001
Relative to the resin (0.1 mmol), 4 equiv DOTA-//7.s(/Bu)ester (2-(4,7,10-tris(2-(t-butoxy)-2- oxoethyl)-l,4,7,10-tetraazacyclododecan-l-yl)acetic acid, 0.400 mmol) were activated with 3.95 equiv HBTU (0.395 mmol) in the presence of 4 equiv DIPEA (0.400 mmol) in 3 mL dry DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized compound 9 and agitated at RT for 3 h. The final products, i.e. P17/P18, were obtained by agitation and subsequent cleavage from the resin and /Bu deprotection within 3 h with a mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water in a ratio of 95:2.5:2.5, respectively. The mixture of solvents from the product was evaporated and the crude compound was dissolved in acetonitrile (ACN) and water in a ratio of 1 : 1 for subsequent reversed-phase high-performance liquid chromatography (RP-HPLC) purification.
Conjugation of Macropa-NCS to P17 and P18 precursor
20 mg of the corresponding amine (Pl 7 precursor or P18 precursor) was dissolved in 200 pL DMF. After the addition of 5.5 mg Macropa-NCS and 10 pL DIPEA, the reaction was stirred for 16 h at the room temperature. The reaction mixture was diluted with water and ACN to a total volume of 4 mL, purified by RP-HPLC and analyzed by ESLMS.
HPLC: Instrument: LATEK P-402; Merck Hitachi L-7420 (UV/VIS signal); Column: Orbit 100 C18 (MZ Analysentechnik), 5 μm, 250x30 mm; Eluents: (A) 95% water + 0.1% TFA, (B) ACN + 0.1% TFA; Gradient: 0-40 min 5-40% B; Flow: 28 mL/min; Temperature: RT; Wavelength:
214 nm; fa (P17-Macropa) = 36.8; fa (P18-Macropa) = 35.0 min; ESLMS: Instrument: Bruker Esquire 600; (P17-Macropa) 1215.3 g/mol; (P18-Macropa) 1221.3 g/mol.
Figure imgf000083_0001
Figure imgf000084_0001
Conjugation of HEHA-NCS to P18 precursor
50 mg of the amine precursor (P18 precursor) was dissolved in 2 mL DMF. After the addition of 25 mg HEHA-NCS ((4-isothiocyanatobenzyl)-l,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid) and 30 pL DIPEA, the reaction was stirred for 16 h at room temperature. After the evaporation of the solvent at 60 °C, the reaction mixture was diluted with water and ACN to a volume of 4 mL, purified by HILIC -HPLC and analyzed by ESI-MS.
Figure imgf000085_0001
HPLC: Instrument: LATEK P-402; Merck Hitachi L-7420 (UV/VIS signal); Column: NUCLEODUR HILIC (Macherey Nagel), 5 μm, 250x21 mm; Eluents: (A) 97% ACN + 0.2% formic acid (FA), (B) water + 0.2% FA; Gradient: 0-40 min 3-50% B; Flow: 15mL/min; Temperature: RT; Wavelength: 214 nm; fa (P18-HEHA) = 29.2 min; ESI-MS: Instrument: Bruker Esquire 600; (P18-HEHA) 1379 g/mol.
Analysis of Pl 7/P18:
P17/P18 were analyzed by RP-HPLC, electrospray ionization mass spectrometry (ESI-MS) as well as ID 'H and 13C nuclear magnetic resonance (NMR), and 2D NMR techniques, viz. correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC). P17 analytics
HPLC: Instrument: LATEK P-402; Merck Hitachi L-7420 (UV/VIS signal); Column: NUCLEODUR HILIC (Macherey Nagel), 5 gm, 250x21 mm; Eluents: (A) 97% ACN + 0.2% formic acid (FA), (B) water + 0.2% FA; Gradient: 0-40 min 3-50% B; Flow: 15mL/min; Temperature: RT; Wavelength: 214 nm; fa (P17) = 24.625 min; ESI-MS: Instrument: Bruker Esquire 600; 1018.2 g/mol; NMR: Instrument: Bruker Avance Neo and Avance III equipped with 5 -mm, inverse-configuration, helium-cooled and 5 -mm, normal-configuration (cryo)probes, respectively, both with z-axis gradient capability at field strengths of 16.45 T and 9.4 T, respectively, operating at 700.13 and 176.05 MHz for 'H and 13C nuclei, respectively, and 400.26, 376.62, 100.64, and 40.56 MHz for 'H, 19F, 13C, and 15N nuclei, respectively; See attachment; Yield: 93 mg (39 %).
P18 analytics
HPLC: Instrument: LATEK P-402; Merck Hitachi L-7420 (UV/VIS signal); Column: NUCLEODUR HILIC (Macherey Nagel), 5 gm, 250x21 mm; Eluents: (A) 97% ACN + 0.2% formic acid (FA), (B) water + 0.2% FA; Gradient: 0-40 min 3-50% B; Flow: 15mL/min; Temperature: RT; Wavelength: 214 nm; fa (P18) = 24.833 min; ESLMS: Instrument: Bruker Esquire 600; 1012.2 g/mol; NMR: Instrument: Bruker Avance Neo and Avance III equipped with 5 -mm, inverse-configuration, helium-cooled and 5 -mm, normal-configuration (cryo)probes, respectively, both with z-axis gradient capability at field strengths of 16.45 T and 9.4 T, respectively, operating at 700.13 and 176.05 MHz for 'H and 13C nuclei, respectively, and 400.26, 376.62, 100.64, and 40.56 MHz for 'H, 19F, 13C, and 15N nuclei, respectively;; Yield: 116 mg (48 %).
Table 1: Analytical information of Pl 7, Pl 8, and PSMA-617.
Figure imgf000086_0001
Radiolabeling with Lu
Radiolabeling of PSMA-617, P17 & P18 with 177Lu was successfully performed;
No-carrier-added lutetium-177 ([177Lu]LuCh; ITM Medical Isotopes GmbH, Germany) was mixed with sodium acetate (0.4 M, pH 4.5). The respective PSMA ligand (1 mM stock solution in DMSO) was added to obtain a molar activity of 1-10 MBq/nmol (dependent on the experiment) and the reaction mixture was incubated for 30 min at 95 °C. Quality control was performed by TLC and HPLC analysis as previously reported. The radiolabeling efficiency was >95% for all tested PSMA ligands.
Co- Crystallization:
Structural studies of PSMA complexes with inhibitors
PSMA was co-crystallized with PSMA-617 and the structure of the complex reported in the past (Benesova, Martina. “Inhibitors of the prostate-specific membrane antigen (PSMA) for PET imaging and endoradiotherapy of prostate cancer.” Dissertation, Ruprecht-Karl s- Universitat Heidelberg, 2016, see also Kopka et al., Journal of Nuclear Medicine September 2017, 58 (Supplement 2) 17S-26S) and crystal structures determined (see below).
Crystallization and data collection:
To this end, diffraction quality crystals were grown using the hanging-drop vapor diffusion method at 288 K. Purified PSMA (10 mg/ml) was mixed with a 50 mM solution of a given inhibitor dissolved in 50 mM Tri-HCl, 150 mM NaCl, pH 8.0, at 9:1 (v/v) ratio. Protein/inhibitor solution was then mixed with the equal volume of mother liquor [34% (v/v) pentaerythritol propoxylate PO/OH 5/4 (Sigma Aldrich), 1 % (w/v) PEG 3350, and 100-mM Tris-HCl, pH 8.0] and 2 pL crystallization droplets equilibrated against 750 pL of the precipitant solution in pregreased Crystalgen SuperClear™ Plates. Rectangular crystals (space group 1222, a = 101 A, b = 130 A, c = 159 A) were flash-frozen directly in liquid nitrogen. Diffraction data were collected from a single crystal using synchrotron radiation at the MX14.1 and MX14.2 beamlines (0.91841 A; BESSYII, Berlin, Germany) equipped with a Pilatus 6M detector (Dectris, Switzerland) at 100 K. Datasets were indexed, integrated and scaled using the XDSAPP interface (Sparta et al., 2016).
Structure refinement
Difference Fourier methods were used to determine structures of PSMA/inhibitor complexes with ligand-free PSMA (PDB code 2OOT and 5O5T) used as a starting model. Iterative refinement and model building cycles were performed using Refmac 5.5. (Koval evskiy et al., 2018) and Coot graphics package (Emsley et al., 2010), respectively. Ligand topologies and coordinates were generated with Acedrg (Long et al., 2017) and inhibitors were fitted into |Fo|— |Fc| electron density maps in the final stages of the refinement. Approximately 1,000 randomly selected reflections were kept aside for cross-validation (Rfree) during the refinement process. Structural findings
Analyses of PSMA complexes revealed that the glutarate-urea docking module of inhibitors interacts with residues lining the Pl pocket and the active-site of PSMA in a manner identical to other urea-based inhibitors described previously. Likewise, the Pl carboxylate function engages side chains of Asn519, Arg534, and Arg536 as noted for analogous PSMA-urea complexes. At the same time, interestingly, marked positional differences are observed for the DOTA chelator and the connecting flexible linker (Fig. 1 IB) together with neighboring PSMA residues (and their interaction patterns).
In the PSMA/617 complex, the wide diameter and partial flexibility of the PSMA entrance funnel allows for the 2-naphtylic function to fold inward and to push the aminohexanoyl group to the opposite wall of the funnel, effectively thus filling its lower portion. To the contrary, the corresponding less lipophilic 3-styryl moiety in P17/P18 is oriented towards the exterior of the protein (Fig. 1 IB) where its phenyl ring is sandwiched between the Lys207 side chain (s-NFL. . .ring center, 3.9 A) and the DOTA function (4.0 A). It shall be noted that variable positioning of distal parts of inhibitors is facilitated by the structural plasticity of the “entrance lid”, the flexible amino acid stretch comprising residues Thr538 - Gly548 of PSMA. In the case of the PSMA/617 complex, the lid adopts the “semi-closed” conformation, and the linker moieties follow the contours of the lid with the 2-naphtyl group engaged in CH/K interactions with Ca carbons of Ser547 (4.5 A) and Gly548 (3.4 A), while the adjacent cyclohexanoyl ring (in the chair conformation) is tightly packed against the phenyl ring of Phe546 with the distance of 5.1 A between the ring centers. For PSMA/P17/P18 complexes, the lid is pushed away from the center of the entrance funnel (up to 6.2 A for corresponding Phe546-Ca carbons) accommodating thus the less compact conformation of the inhibitor linker (Fig. 1 IF).
Finally, the Fo-Fc electron density peaks are missing for the DOTA chelator of 617, and this observation is consistent with the absence of intermolecular interactions with PSMA and ensuing positional flexibility outside the entrance funnel of PSMA. On the other hand, the DOTA function is well resolved in Fo-Fc maps of PSMA/P17/P18 complexes, where its position is stabilized via an array of CH/K interactions between the methylene groups of the central 12-membered tetraazacyclododecane ring and the indole group of the Trp541 side chain of the entrance lid (Fig. 11 A, G).
In vitro IC50 determination
Inhibitory potency studied compounds was determined using a fluorescence-based assay described previously (DOI: 10.1021/acs.jcim.2c01269). Briefly, 20 pM recombinant human PSMA was preincubated with 3-fold dilution series of inhibitors in 50 mM Tris-HCl, 150 mM NaCl, 0.001 % C12E8 for 10 min at 37°C. The reaction was initiated by the addition of 100 nM substrate in the total volume 50 pL. Following 15 min incubation at 37 °C, the reaction was stopped by the addition of 50 pL of 0.1 % trifluoroacetic acid (TFA) in 5% acetonitrile. Reaction mixtures were analyzed by RP-HPLC (Shimadzu, HPLC Prominence system) on a Kinetex 2.6 pm XB-C18 100 A column with fluorescence detection (ZEX/kEM = 492/516). HPLC separation was performed over an 11-min linear gradient from 20 to 50 % of eluent B (0.1% (v/v) TFA in acetonitrile) at a flow rate of 0.6 mL/min. The mobile phase A was 0.1% (v/v) TFA in water. The non-inhibited reaction was assigned to 100%, and the reaction without an enzyme was defined as 0%. The data were fitted using the GraphPad Prism software and IC50 values were calculated from the inhibition curves of three independent experiments using a nonlinear regression protocol.
Potency of studied inhibitors against recombinant human PSMA was determined in vitro using an HPLC-based assay with fhioresceine-y-Glu-Glu as a substrate and calculated IC50 values were 145, 344, and 128 pM for 617, P17, and P18, respectively. The subnanomolar inhibitory potencies are virtually identical for the parent 617 and P17/P18 derivatives and reflect tight binding to PSMA via extensive contacts as observed in our X-ray structures of PSMA/inhibitor complexes (above).
It is interesting to note that affinities of metal-loaded P17/P18 inhibitors for PSMA are lower compared to metal-free counterparts and trends in binding affinities correlate with the metal coordination via carboxylic pendants (binding affinity better for natGa involving only two pendants than for natBi/natLa involving all four) as well as size of the metal (binding affinity worse for natLa with 195 ppm than for natBi with 160 ppm and natGa with 130 ppm) —> IC50 L > IC50 [natGa]-L > [natBi]-L > [natLa]-L. These finding are in line with X-ray structures of PSMA/inhibitor complexes, where the metal-free DOTA chelator interacts with PSMA and increases thus inhibitor potency via avidity effects while metal-loaded DOTA is expected to lack such interactions.
IC50 values performed via PSMA cell-based assay of Pl 7, P18 and PSMA-617 on PSMA+ cell lines LNCaP, C4-2, and PC3-PIP (n=3) were determined.
Cell-binding affinity assay
The binding affinities of PSMA-617, P17 and Pl 8 were determined using a competitive assay with [177LU]LU-PSMA-617. All three compounds were incubated at 12 different concentrations (0, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 500, 1000 and 5000 nM) with 0.75 nM [177Lu]Lu-PSMA-617 on either LNCaP (PSMA+), C4-2 (PSMA+), PC3-PIP (PSMA+) or PC3-flu (PSMA-) cells. After 45 min incubation at the RT, the activity was removed and cells were washed twice with 100 pL and once with 200 pL ice-cold PBS. The activity specifically accumulated in the cells was measured in a gamma counter (Cobra Autogamma B5003, Canberra, Packard; Frankfurt, Germany). The data were fitted using a non-linear regression algorithm (GraphPad Prism Software, version 8) to calculate 50% inhibitory concentrations (IC50 values). The experiment was repeated in three independent assays performed in a quadruplicate and the data are presented as the average ± SD. Statistical analysis using multiple unpaired t-test (GraphPad Prism Software, version 8) was pursued and a p- value of <0.05 was considered statistically significant.
Internalization rate of [177Lu]Lu-P17, [177Lu]Lu-P18 and [177Lu]Lu-PSMA-617 on PSMA+ cell line PC3-PIP (n=4) was determined. The internalization rates of [177Lu]Lu-PSMA-617, [177Lu]Lu-P17 and [177Lu]Lu-P18 were tested on either PC3-PIP (PSMA+) or PC3-flu (PSMA-) cells. The cells were seeded in poly- L-ly sine-coated plates 24 h before the experiment. The cells were incubated for 45 min at 37 °C with 30 nM [177Lu]Lu-P17, [177Lu]Lu-P18 or [177Lu]Lu-PSMA-617 in 250 pL Opti-MEM (Gibco). Moreover, another set of cells was treated analogically while the PSMA-inhibitor 2- (phosphonomethyl)pentane- 1,5 -dioic acid (2-PMPA; 100 mM, 500 pM/well) was added in order to subtract the non-specific binding. The activity was removed and cells were washed three times with 1 mL ice-cold PBS and the surface-bound radioactivity was removed by incubating the cells twice with 500 pL glycine buffer (50mM; pH 2.8) for 5 min. After washing the cells once with 1 mL ice-cold PBS, the internalized fraction was determined by subsequent cells lysis with 500 pL NaOH (0.3M; pH 14). The collected glycine and hydroxide fractions were measured in a gamma counter (Cobra Autogamma B5003, Canberra, Packard; Frankfurt, Germany) and calculated as percentage of total applied activity specifically bound on either cells surface or internalized inside the cells. The experiment was repeated in three independent assays performed in a triplicate and the data are presented as the average ± SD. Statistical analysis using multiple unpaired t-test (GraphPad Prism Software, version 8) was pursued and a p- value of <0.05 was considered statistically significant.
Biodistribution studies of [177Lu]Lu-P17, [177Lu]Lu-P18 and [177Lu]Lu-PSMA-617 were performed in PC3-PIP/-flu tumor-bearing Balb/c nude mice (n=3) 1 and 2 h post-injection.
Tumor-bearing mice were intravenously injected with either [177Lu]Lu-PSMA-617, [177Lu]Lu- P17 and [177Lu]Lu-P18. The mice were sacrificed at 1 h, 2 h and 24 h post-injection (p.i.). Groups of 3 mice were used for each time-point. Selected tissues and organs were collected, weighed, and measured in a gamma counter (Cobra Autogamma B5003, Canberra, Packard; Frankfurt, Germany). The results were calculated as percentage of the injected activity per gram of tissue mass (% lA/g) and the data are presented as the average ± SD.
Table 2: IC50 values determined via GCPILbased enzymatic assay.
Figure imgf000090_0001
Figure imgf000090_0002
Table 3: IC50 values determined via PSMA cell-based assay.
Figure imgf000091_0001
Table 4: Internalization rate.
Figure imgf000091_0002
Table 5. Biodistribution data obtained in PC3-PIP and PC3-flu tumor-bearing balb/c nude mice at 1, 2 and 24 h after injection of [177Lu]Lu-P17.
Figure imgf000091_0003
Salivary glands 0.23 ± 0.04 0.10 ± 0.02 0.03 ± 0.01
Figure imgf000092_0001
Figure imgf000093_0001

Claims

1. A compound of formula (1)
Figure imgf000094_0001
or a pharmaceutically acceptable salt or solvate thereof, wherein R1 is H or -CH3, preferably H, wherein R2, R3 and R4 are independently of each other, selected from the group consisting of -CO2H, -SO2H, -SO3H, -OSO3H, -PO2H2, -PO3H2, -OPO3H2, - CONH2 and -CONHOH, wherein L is a suitable linker, linking A and NH, preferably L is a linking group selected from the group consisting of -(Q3)q-NH-C(=O)-, -(Q3)q-C(=O)-, -(Q3)q-C(=S)-, -(Q3)q- NH-C(=S)-, wherein Q3 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is 0 or 1, and wherein n is 0 or 1 wherein XAA1 is an amino acid moiety having the structure (A)
Figure imgf000094_0002
with Q1 being a residue comprising an optionally substituted styryl moiety, said residue preferably having the structure -(CHR5)m-CH=CH-Ph, where m equals 0, 1, 2, or 3, each R5, independently of others, is selected from the group consisting of H, alkyl and aryl, and Ph is phenyl optionally substituted with one to five substituents selected from alkyl and aryl groups,
Q2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and
A is a chelator residue derived from a chelator selected from the group consisting of
1.4.7.10-tetraazacyclododecane-N,N',N",N "'-tetraacetic acid (= DOTA), N,N"-bis[2- hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid, 1,4,7- triazacyclononane-l,4,7-triacetic acid (= NOTA), 2-(4,7-bis(carboxymethyl)- 1,4,7- triazonan-l-yl)pentanedioic acid (=NODAGA), 2-(4,7,10-tris(carboxymethyl)-
1.4.7.10-tetraazacyclododecan-l-yl)pentanedioic acid (=DOTAGA), 1,4,7- triazacyclononane phosphinic acid (=TRAP), l,4,7-triazacyclononane-l-[methyl(2- carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (=NOPO), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene-3,6,9-triacetic acid (= PCTA), l,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- methylperhydro- 1 ,4-diazepine (=AAZTA), 1 ,4,7, 10-tetra-azacyclododecan- 1,4,7,10- tetra-azidoethylacetic acid (=DOTAZA), Nl-(5-aminopentyl)-Nl-hydroxy-N4-(5-(N- hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4- oxobutanamido)pentyl)succinamide (=DFO), Nl-[5-(Acetylhydroxyamino)pentyl]- N26-(5-aminopentyl)-N26,5, 16-trihydroxy-4, 12, 15,23 -tetraoxo-5,11, 16,22- tetraazahexacosanediamide (=DFO*), oxo-DFO*, N,N'-bis[(6-carboxy-2- pyridil)methyl]-4,13-diaza-18-crown-6 (macropa), 4-Amino-6-((16-((6- carboxypyridin-2-yl)methyl)-l,4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7- yl)methyl)picolinic acid (=Macropa-NH2), 2,2'-(ethane-l,2-diylbis(((8- hydroxyquinolin-2-yl)methyl)azanediyl)) diacetic acid (=H4octox), 6,6'-((9-hydroxy- l,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7- diyl)bis(methylene))dipicolinic acid (=H2bispa), dimethyl 9-hydroxy-3,7-bis((8- hydroxyquinolin-2-yl)methyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-l,5- dicarboxylate (=H2bispox), 6,6'-(((pyridine-2,6-diylbis(methylene))- bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid (=H4py4pa), Diethylenetriaminepentaacetic acid (=DTPA), Trans-cyclohexyl- diethylenetriaminepentaacetic acid (=CHX-DTPA), 1,4,7, 10-tetraazacyclododecane- 1,4,7-triacetic acid (=DO3A), l-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (=oxo-Do3 A), 1,4,7, 10-Tetrakis(carbamoylmethyl)- 1,4,7, 10-tetraazacyclododecane
(=DOTAM), N,N'-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N'-diacetic acid (=H40ctapa), 1,4,7,10,13,16-hexaazacyclohexadecane- 1,4,7,10,13,16-hexaacetic acid (=HEHA), 1,4,7,10,13 -pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (=PEPA), cyclam-based ligands with N-methyl, N-(4-aminobenzyl), N-phosphinate- and phosphonate-based substituents.
2. The compound of claim 1, wherein the compound has the structure (la),
Figure imgf000095_0001
preferably, the structure (laa)
Figure imgf000096_0001
wherein R5, R6, R7, R8, R9, and R10 are, independently of each other, selected from the group consisting of, H, alkyl and aryl, preferably H and alkyl, more preferably H and methyl, most preferably H.
3. The compound of claim 1 or 2, wherein A is a chelator residue having a structure selected from the group consisting of
Figure imgf000096_0002
Figure imgf000097_0001
4. The compound of any of one of claims 1 to 3, wherein R3, R2 and R4 are -CO2H and R1 is H.
5. The compound of any one of claims 1 to 4, wherein Q2 is
Figure imgf000097_0002
preferably
Figure imgf000097_0003
6. The compound of any one of claims 1 to 5 having a structure selected from the group consisting of
Figure imgf000098_0001
Figure imgf000099_0001
and
Figure imgf000100_0001
more preferably having the structure
Figure imgf000100_0002
Figure imgf000101_0001
7. Complex comprising
(c) a radionuclide, and
(d) the compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt or solvate thereof.
8. The complex of claim 7, wherein, the radionuclide is selected from the group consisting 18F, 89Zr, 43Sc, 44Sc, 47Sc, 45Ti, 55Co, mIn, 86Y, 90Y, 66Ga, 67Ga, 68Ga, 177Lu, "mTc, 60Cu, 61Cu, 62Cu, 64Cu, 66Cu, 67Cu, 149Tb, 152Tb, 155Tb, 153Sm, 161Tb, 166Ho, 153Gd, 155Gd, 157Gd, 211At, 213Bi, 225 Ac, 230U, 223Ra, 224Ra, 165Er, 52Fe, 59Fe, 186Re, 188Re, 227Th, 133La, 72 As and radionuclides of Pb.
9. Method for preparing a compound of formula (1) according to any one of claims 1 to 6 the method comprising:
(aa) attaching Fmoc-LysfNPG1 )-OH to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG1 is a suitable protecting group, preferably being orthogonal to Fmoc and 2-CT linker of the resin, more preferably Alloc,
(bb) reacting H-G1U(OPG2)-OPG2 with 1,1 ’ -carbonyldiimidazole in the presence of a suitable base, thereby forming the activated Glu compound of formula (II)
Figure imgf000102_0001
wherein PG2 is a suitable protecting group, preferably being orthogonal to Fmoc and Alloc, more preferably tBu
(cc) reacting the compound of formula (II) with the Lys modified resin according to step (aa) and removing PG1, thereby forming the modified resin
Figure imgf000102_0002
(dd) reacting the modified resin according to (cc) with Fmoc-(XAA1)-OH, and removing the Fmoc group thereby forming the modified resin
Figure imgf000102_0003
(ee) reacting the modified resin according
Fmoc — NH— CH
Figure imgf000102_0004
and removing the Fmoc group,
(ff) linking the free amino group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L, (gg) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
10. Method for preparing a compound of formula (1) according to any one of claims 1 to 6, the method comprising:
(a) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(b) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000103_0001
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu,
(c) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin
Figure imgf000103_0002
preferably
Figure imgf000103_0003
(d) reacting the modified resin according to (c) with Fmoc-(XAAi)-OH, and removing the Fmoc group thereby forming the modified resin
Figure imgf000104_0001
preferably
Figure imgf000104_0002
reacting the modified resin according to (d) with
Fmoc — NH— CH2— Q21 and removing the Fmoc group,
(f) linking the free amino group with the, optionally protected, chelator residue A, wherein optionally A and the free amino group are linked via the linking group L,
(g) cleaving the compound from the resin and removing all remaining protecting groups thereby forming a compound of formula (1).
11. Method according to claim 10, wherein no Alloc protecting group is used.
12. Method for preparing a resin bound PSMA binding moiety having the structure
Figure imgf000105_0001
preferably
Figure imgf000105_0002
as intermediate product for the solid phase synthesis of PSMA ligands, preferably for the synthesis of a PSMA ligand according to embodiment 1 to 11, the method comprising the steps,
(a) attaching Fmoc-Glu(OPG2)-OH to a suitable resin, preferably a 2-Chloro-trityl resin (2-CT), and removing the Fmoc protecting group, wherein PG2 is a protecting group being orthogonal to Fmoc, preferably tBu,
(b) reacting H-Lys(Fmoc)-OPG4 with 1,1’ -carbonyldiimidazole in the presence of a suitable base, thereby forming a compound of formula (II)
Figure imgf000105_0003
FmocNH wherein PG4 is a protecting group being orthogonal to Fmoc, preferably tBu,
(c) reacting the compound of formula (II) with the Glu modified resin according to step (a) and removing the Fmoc group, thereby forming the modified resin.
13. A pharmaceutical composition comprising a compound of any one of claim 1 to 6 or a complex of claim 7 or 8.
14. A compound of any one of claim 1 to 6 or a complex of claim 7 or 8 for use in medicine and/or in diagnostics.
15. A compound of any one of claim 1 to 6 or a complex of claim 7 or 8 for use in diagnosing, treating and/or preventing PSMA expressing cancer, in particular prostate cancer and/or metastases thereof.
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