WO2018233798A1 - Novel psma-binding agents and uses thereof - Google Patents

Novel psma-binding agents and uses thereof Download PDF

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Publication number
WO2018233798A1
WO2018233798A1 PCT/EP2017/000717 EP2017000717W WO2018233798A1 WO 2018233798 A1 WO2018233798 A1 WO 2018233798A1 EP 2017000717 W EP2017000717 W EP 2017000717W WO 2018233798 A1 WO2018233798 A1 WO 2018233798A1
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Prior art keywords
psma
compound
acid
compound according
hydrocarbyl
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PCT/EP2017/000717
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French (fr)
Inventor
Martina Benesova
Cristina MÜLLER
Christoph UMBRICHT
Roger Schibli
Konstantin Zhernosekov
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ITM Isotopen Technologien München AG
Paul Scherrer Institut
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Application filed by ITM Isotopen Technologien München AG, Paul Scherrer Institut filed Critical ITM Isotopen Technologien München AG
Priority to PCT/EP2017/000717 priority Critical patent/WO2018233798A1/en
Priority to EP18725856.1A priority patent/EP3630204A1/en
Priority to AU2018274184A priority patent/AU2018274184A1/en
Priority to RU2019141963A priority patent/RU2787105C2/en
Priority to CN201880034324.3A priority patent/CN110740757B/en
Priority to PCT/EP2018/063734 priority patent/WO2018215627A1/en
Priority to JP2019562312A priority patent/JP7340459B2/en
Priority to US16/616,744 priority patent/US11629201B2/en
Priority to KR1020197038065A priority patent/KR20200009091A/en
Priority to CA3060143A priority patent/CA3060143A1/en
Publication of WO2018233798A1 publication Critical patent/WO2018233798A1/en
Priority to ZA2019/06832A priority patent/ZA201906832B/en

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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
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • 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
    • A61K51/0495—Pretargeting

Definitions

  • the present invention relates to novel compounds and radiolabeled complexes comprising a chelating agent, a PSMA-binding entity and an albumin-binding entity connected via suitable linkers and spacers, which are envisaged for use as diagnostic and/or therapeutic radiopharmaceuticals.
  • the compounds and complexes according to the invention lend themselves as (theragnostic) tracers, imaging agents and therapeutic agents for detecting PSMA-expressing target cells and tissues and treating and diagnosing cancer.
  • Prostate cancer is the leading cancer in the US and European population. 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. According to the American Cancer Society, approximately 1 61 ,000 new cases of prostate cancer are diagnosed each year in USA. The 5-year survival rate of patients with stage IV metastatic prostate cancers is only about 29%.
  • Tumor antigens may be broadly classified into two categories based on their expression pattern: Tumor-Specific Antigens (TSA), which are present only on tumor cells and not on non-malignant cells and Tumor-Associated Antigens (TAA), which are present on some tumor cells and also non- malignant cells.
  • TSAs typically emerge as a result of the mutation of protooncogenes and tumor suppressors which lead to abnormal protein production, whereas TAA expression is general ly caused by mutation of other genes unrelated to the tumor formation.
  • Proteinaceous binding agents or small molecule drugs carrying visualizable labels and specifically recognizing such tumor markers are typically employed for diagnosing and imaging cancers under non- invasive conditions.
  • PSMA prostate-specific membrane antigen
  • FOLH1 folate hydrolase I
  • FOLH1 folate hydrolase I
  • the PSMA gene is located on the short arm of chromosome 1 1 and functions both as a folate hydrolase and neuropeptidase.
  • GCPII glutamate carboxypeptidase II
  • PSMA glutamate carboxypeptidase II
  • PSMA is (i) mainly restricted to the prostate (although is also detected in lower amounts in the neovasculature of numerous other solid tumors, including bladder, pancreas, lung, and kidney cancers, but not in normal vasculature), (ii) abundantly expressed as protein at all stages of prostate cancer (in amounts of up to 1 0 6 PSMA molecules per cancer cell) (iii) presented at the cell surface but not shed into the circulation, and (iv) associated with enzymatic or signaling activity. Moreover, PSMA expression is further up-regulated in poorly differentiated, androgen-i nsensitive or metastatic cancers and the expression usually correlateds with disease progression. The unique expression of PSMA makes it an important marker of prostate cancer (and a few other cancers as well).
  • PSMA represents a large extracel lular target for imaging agents.
  • PSMA is internalized after ligand binding and, thus, it is not only an excel lent target for targeted radionuclide therapy (using particle-emitting radionuclides) but also for other therapeutic strategies including the tumor cel l-specific delivery of immunotoxins, retargeting of immune cells, pro-drug activation, PSMA vaccines, and plasmid DNA and adenoviral immunizations. Because of low expression levels in healthy tissue, PSMA has additional ly the potential for high-dose therapy, with minimized side effects.
  • PSMA targeting agents capable of binding to the extracellular domain of PSMA were developed for PET/CT and SPECT/CT imaging, including radiolabeled N-[N-[(S)- 1 ,3-dicarboxypropyl]carbamoyl]-S-[1 1 C]methyl-l-cysteine (DCFBC) and several urea-based peptidomimetic PSMA-inhibitors (cf. Bouchelouche et al. Discov Med. 201 0 Jan; 9(44): 55- 61 ), including MIP-1 095 (H illier et al. Cancer Res.
  • the ligands developed by Kelly et al. comprise a >(iodophenyl)butyric acid entity for HSA binding and an urea-based PSMA binding entity.
  • radiotherapeutic iodine ( 131 l) is covalently attached to the HSA binding moiety, which is in turn directly connected to the PSMA binding entity via a hydrocarbyl chain.
  • the evaluated compounds are considerably limited in terms of the applied radionuclide which is limited to iodine. Further, no improved internal ization/uptake in target cells was demonstrated for the evaluated compounds.
  • Another approach was followed by Choy et al. Theranostics 201 7; 7(7):1 928-1 939, who evaluated 177 Lu-labeled phosphoramidate-based PSMA inhibitor with an albumin- binding entity.
  • a DOTA chelator complexing the 177 Lu radionuclide was ether-linked to the irreversible PSMA inhibitor CTT1 298 (EP 2970345 A1 ).
  • Phosphoramidate-based PSMA binding motive exhibits only poor stabi lity, especial ly at elevated temperatures (elevated temperatures under extended acidic conditions lead to hydrolysis of phosphoramidate P-N bond), which are required for the coordinative radiolabeling reaction via chelators such as DOTA. Therefore a direct radiolabeling reaction cannot be applied and a multi-step pre-labeling approach has to be used.
  • Lu-DOTA-azide as precursor should be prepared; subsequently the precursor has to be coupled to a dibenzocyclooctyne- derivatized PSMA motive.
  • elaborate HPLC purification of the coupled compound must be undertaken; reformulation with evaporation (under N2 atmosphere) of the HPLC- eluent and dissolving in a physiological medium need to be performed. This procedure is likely not possible for a clinical application when high activities are being produced. Pre- clinical biodistribution data demonstrate poor performance of the radiolabeled agent especially regading tumour-to-kidney ratios which did not exceed far above 1 .
  • the term herein is a particular embodiment of the term herein”, wherein any other non-stated member, integer or step is excluded.
  • the term herein encompasses the term beforeconsist of".
  • the term Lincolncomprising thus encompasses flickincluding” as well as searchingconsisting” e.g., a composition instructcomprising" X may consist exclusively of X or may include something additional e.g., X + Y.
  • the term termed herein refers to residues of hydrocarbon groups, i.e., hydrocarbon chain radicals, preferably independently selected from the group alkyl, alkenyl, alkynyl, aryl and aralkyl.
  • the term “excellentalkyl” comprises linear diligencestraight-chain”), branched and cyclic chain radicals having 1-30 carbon atoms, preferably 1-20, 1-15, 1-10, 1-8, 1-6, 1-4, 1-3 or 1-2 carbon atoms.
  • the term “excellentCi-i 2 alkyl” refers to a hydrocarbon radical whose carbon chain is straight-chain or branched or cyclic and comprises 1 to 12 carbon atoms.
  • alkyl residues are methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl or triacosyl, including the various branched-chain and/or cyclic isomers thereof, e.g.
  • Cyclic alkyl isomers are also referred to as dominantcycloalkyl" herein to refer to saturated alicyclic hydrocarbons comprising 3 ring carbon atoms.
  • Substituted" linear, branched and cyclic alkyl groups are generally also encompassed by the term.
  • the term further includes counselheteroalkyl", referring to alkyl groups wherein one or more C-atoms of the carbon chain are replaced with a heteroatom such as, but not limited to, N, O, and S.
  • the term further includes amongheterocyclyl" or possiblyheterocycloalkyl", referring to non-aromatic ring compounds containing 3 or more ring members, of which one or more ring carbon atoms are replaced with a heteroatom such as, but not limited to, N, O, and S.
  • Heterocyclyl groups encompass unsaturated, partially saturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups.
  • Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl,
  • Heterocyclyl groups may be substituted or unsubstituted.
  • Representative substituted heterocyclyl groups may be monosubstituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above.
  • the term taucyclic includes the term FAQ, ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ,
  • the term “suitalkenyl” as employed herein comprises linear, branched and cyclic chain 1 0 radicals having 2-30 carbon atoms, preferably 2-20, 2-1 5, 2-1 0, 2-8, 2-6, 2-4, or 2-3 carbon atoms, including at least one carbon-to-carbon double bond.
  • Specific examples of frequentlyalkenyl” groups are the various alkenic unsaturated equivalents of those given with respect to alkyl groups, named after the conventions known to the person ski lled in the art, depending on the number and location of carbon-to-carbon double bond or bonds, e.g.
  • alkenyl groups preferably contain at least 1 , more preferably at least 2, 3, 4, 6, 7, 8, 9, 1 0, 1 1 , 1 2, 1 3, 14, 1 5, or 1 6 double bonds, wherein a double bond is preferably located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 12, 1 3, 1 4, 1 5, 1 6, 1 7, 1 8, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 of the hydrocarbyl chain.
  • Alkenyl groups may be substituted or unsubstituted.
  • the term “suitalkynyl” as employed herein comprises straight, branched and cyclic chain radicals having 2-30 carbon atoms, preferably 2-20, 2-1 5, 2-1 0, 2-8, 2-6, 2-4, or 2-3 carbon atoms, including at least one carbon-to-carbon triple bond.
  • ⁇ ⁇
  • alkynyl ⁇
  • alkynyl groups preferably contain at least 1 , more preferably at least 2, 3, 4, 6, 7, 8, 9, 1 0, 1 1 , 1 2, 1 3, 14, 1 5, or 1 6 triple bonds, wherein a double triple bond is preferably located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 12, 1 3, 14, 1 5, 1 6, 1 7, 30 1 8, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 of the hydrocarbyl chain.
  • Alkynyl groups may be substituted or unsubstituted.
  • the term “bigaryl” refers to monocyclic or polycyclic or fused polycyclic aromatic ring systems.
  • the term includes monocyclic or polycyclic or fused polycyclic aromatic possiblyheteroaryl" ring systems wherein at least one carbon atom of the ring system is substituted by a heteroatom.
  • the terms “clientaryl” and “contentheteroaryl” refers to groups having 3-30 carbon atoms., such as 3-10, in particular 2-6 carbon atoms.
  • aralkyi group as defined herein, the aralkyi group is bonded to another moiety of the compounds or conjugates of the invention via the alkyl group as exemplified by a benzyl group.
  • the term graspheteroatom includes N, O, S and P, preferably N and O.
  • substituted refers to a hydrocarbyl group, as defined herein (e.g., an alkyl or alkenyl group) i n which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms.
  • substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom.
  • a substituted" group will be substituted with one or more substituents, unless otherwise specified.
  • a substituted group is substituted with 1 , 2, 3, 4, 5, or 6 substituents.
  • substituent groups i n include: halogens (i.e., F, CI, Br, and I); hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls ( oxo ); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups;
  • the present invention provides novel plasma protein-binding PSMA ligands with improved tumor targeting properties and favorable pharmacokinetic profi les.
  • the term pharmacoki netics preferably includes the stabi lity, bioavailability, absorption, biodistribution, biological half-life and/or clearance of a therapeutic or diagnostic agent in a subject.
  • the present inventors provided novel conjugates by covalently coupling a PSMA- peptidomimetic urea-based binding entity via suitable spacers and linkers to a a chelator capable of complexing therapeutic/diagnostic radionuclides on the one hand, and a human serum albumin (HSA) binding entity on the other hand.
  • HSA human serum albumin
  • the spacer and linker groups connecting the binding entities and chelator were found to be crucial for the targeting and pharmacokinetic properties of the resulting conjugates.
  • the novel conjugates preferably exhibit superior and specific cellular uptake and internalization characteristics.
  • the inventors demonstrated that the HSA binding entity advantageously effected (1 ) compartmentalization of the conjugates in the blood (where off-target effects in healthy tissues are limited, without compromising access to the tumor vasculature), (2) extended blood clearance, and (3) increased tumor uptake and retention (by increasing the number of passes through the tumor bed). Introduction of a HSA binding entity thereby advantageously improves biodistribution and, eventually, therapeutic efficacy of the inventive compounds.
  • the conjugates provided herein advantageously exhibit an increased tumor uptake as compared to other PSMA ligands known in the art.
  • the conjugates' favourable tumor uptake characteristics in particular al low reducing the administered activity to achieve the desired dose for a therapeutic effect or sufficient uptake allowing imaging (diagnosis).
  • the conjugates are commonly provided in the form of radiolabeled complexes with the chelator complexing a therapeutic and/or diagnostic radionuclide (often a metal isotope).
  • the inventive conjugates are thus promising theragnostic agents with optimal characteristics both for nuclear imaging and endoradiotherapy.
  • the novel PSMA l igands according to the invention thus include a first terminal group (a chelating agent), a second terminal group (an albumin binding entity) and a third terminal group (a PSMA binding entity) that are covalently connected or linked to each other via appropriate linkers or spacers.
  • the present invention relates to a compound of General Formula (1 ):
  • Abm is an albumin binding entity, preferably as defi ned herein,
  • Pbm is a PSMA binding entity, preferably as defined herein,
  • the spacer comprises at least one C-N bond
  • the -CH- group i n General Formula (1 ) is a "branching point" connecting the PSMA binding entity (Pbm) and the albumin binding entity (Abm), or pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof.
  • D, Abm, Pbm, Linker and Spacer are preferably defined as described herein.
  • the present invention provides compounds according to General Forrr (D(i) or (D(ii):
  • Abm is an albumin bi nding entity, preferably as defined herein
  • Pbm is a PSMA bi nding entity, preferably as defi ned herein
  • D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2 -(4,7, 1 0-tris(carboxymethyl)-1 ,4,7,1 0- tetraazacyclodo
  • X is each independently selected from O, N, S or P,
  • R 6 and R 7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl
  • R 3 , R 4 and R 5 are each independently selected from -COH, -C0 2 H, -SO2H, - SO 3 H, -SO 4 H, -PO 2 H, -PO 3 H, -PO 4 H 2 , -C(0)-(G-Go)alkyl, -C(O)-O(G-G 0 )alkyl, - C(0)-NHR 8 , or -C(0)-NR 8 R 9 ' wherein R 8 and R 9 are each independently selected from H, bond, (G -Go)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(Ci-Cio)alkylene, -(CH 2 ) P -NH, -(CH 2 ) p -(Ci-Ci 0 )alkyene, -(CH 2 ) P -NH
  • the spacer comprises at least one C-N bond
  • the linker is characterized by General Formula (6) as defined herein, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0,
  • the inventive conjugates are ligands exhibiting affinity towards both PSMA and HSA.
  • ligand refers to a compound capable of interacting with (targeting, binding to) a target (here: PSMA or HSA).
  • the inventive conjugates may also be defined functional ly as "PSMA targeting agents".
  • PSMA targeting agents Preferably, "ligands" are capable of selectively binding to their target.
  • selective binding means that a compound binds with a greater affinity to its intended target than it binds to another, non-target entity.
  • Binding affinity is the strength of the bi nding interaction between a ligand (e.g. a smal l organic molecule, protein or nucleic acid) to its target/binding partner. Binding affinity is typically measured and reported by the equilibrium dissociation constant (K D ), a ratio of the "off-rate” (k 0 ff) and the “on-rate” (k on ), which is used to evaluate and rank order strengths of bi molecular interactions.
  • K D equilibrium dissociation constant
  • the term "selectively binding” preferably means that a ligand binds to its intended target with a K D that is lower than the K D of its binding to another, non-target entity.
  • binding affinity and dissociation constants such as ELISA, gel-shift assays, pull-down assays, equi librium dialysis, analytical ultracentrifugation, surface plasmon resonance, and spectroscopic assays.
  • the K D for binding of the PSMA binding entity (HSA binding entity) to a non-target entity may be at least 1 .5-fold, preferably at least 2-, 3-, 5-, 10-, 1 5-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 1 00- 200-, 300-, 400-, 500-, 750-, or 1000-fold the K D for binding of said conjugate or moiety to human PSMA (HSA).
  • the conjugates bind to PSMA with high binding affinity with K D values in the nanomolar (nM) range and with moderate affinity to HSA in the micromolar range ( ⁇ (micromolar)).
  • nM nanomolar
  • ⁇ micromolar
  • the inventive conjugates may exhibit a higher binding affinity towards PSMA than towards HSA.
  • the inventive conjugates comprise an (additional -as compared to known PSMA ligands) albumin binding entity (also referred to as an "albumin binding moiety") as described herein, which is preferably capable of selectively binding to human serum albumin (HSA).
  • HSA human serum albumin
  • Abm is an albumin binding entity
  • D is a chelator, preferably selected from 1 ,4,7,1 O-tetraazacyclododecane- 1 ,4,7,1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOT A), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2 -(4,7, 1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1
  • DTPA Diethylenetriaminepentaacetic acid
  • X is selected from O, N, S or P,
  • R 3 , R 4 and R 5 are each independently selected from -COH, -CC1 ⁇ 2H, -SO2H, - SO 3 H, -SO4H, -PO 2 H, -PO 3 H, -P0 4 H 2 , -C(O)-(Ci-Ci 0 )alkyl / -C(O)-O(Ci-Ci 0 )alkyl, - C(0)-NHR 8 , or -C(0)-NR 8 R 9, wherein R 8 and R 9 are each independently selected from H, bond, (Ci-Cio)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(Ci-Cio)alkylene, -(CH 2 ) P -NH, -(CH 2 )p-(Ci-C 10 )alkyene, -(
  • the spacer comprises at least one C-N bond
  • the linker is characterized by General Formula (6) as defined herein, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0, or a pharmaceutical ly acceptable salt, ester, solvate or radiolabeled complex thereof.
  • the albumin binding entity may be any albumin binding entity. Particularly preferred albumin binding entities are described herein below.
  • the albumin binding entity may preferably bind non-covalently to serum albumin, preferably HSA, typically with a binding affinity of less than about 100 ⁇ (micromolar), e.g. of about 3 ⁇ (micromolar) to 50 ⁇ (micromolar).
  • Human Serum Albumin is the most abundant protein in human plasma and constitutes about half of serum protein.
  • Human Serum Albumin or "HSA” as used herein preferably refers to the serum albumin protein encoded by the human ALB gene. More preferably, the term refers to the protein as characterized under UniProt Acc. No. P02768 (entry version 240, last modified May 1 0, 201 7, or functional variants, isoforms, fragments or (post-translationally or otherwise modified) derivatives thereof.
  • the albumin binding entity (Abm) of the inventive conjugates preferably extends circulation half-life of the conjugates, and effects compartmentalization of the inventive conjugates in the blood and improved delivery to the PSMA-expressing (tumor) target cel ls or tissues, resulting in increased tumor: non-target ratios for PSMA expressing normal (non-tumorous) organs (like kidneys, lacrimal glands, and salivary glands).
  • the albumin binding entity is thus envisaged to confer improved pharmacokinetic properties to the inventive conjugate, preferably without interfering with (reduci ng or abolishing) the desired function of the chelating agent and the PSMA binding entity.
  • typical albumin binding entities in accordance with the present invention may preferably comprise linear and branched lipophilic groups comprising 1-40 carbon atoms and a distal acidic group.
  • Suitable albumin binding entities are inter alia described in US 2010/172844 A1, WO 2013/024035 A1 and WO 2008/053360 A2, which are incorporated by reference in their entirety herein.
  • the albumin binding entity is preferably characterized by General Formula (2):
  • R 6 and R 7 are each independently selected from H or branched, unbranched or cyclic G.12 hydrocarbyl, and X is selected from O, N, P or S.
  • R 1 and R 2 may be in ortho-, meta or para-position.
  • said cyclic structure is preferably a linear or branched hydrocarbyl chain of 3-1 2, more preferably 3-1 0, even more preferably 3-9, 3-8, 3-7, 3-6, 3-5, 3-4 or 4 carbon atoms bonded at two positions to the phenyl ring, i.e. forming two bonds to said phenyl ring, such as to form a ring structure fused to said phenyl ring.
  • said cyclic structure may be selected from (substituted or unsubstituted) adamantyl.
  • said two bonds are preferably situated at the meta (3-) and para (4-) positions, at the ortho (2-) and meta positions or at the ortho and para positions of said phenyl ring.
  • Said cyclic structure is optionally interrupted by up to 2, preferably 1 or none heteroatoms.
  • said cyclic structure may be a C4 chain fragment (1 ,4-diradical) linked by its 1 - and 4- atoms to said phenyl ring to form a six-membered ring fused to said phenyl ring, preferably at the meta and para positions of said phenyl ring, i.e., preferably forming a meta- and para-fused six-membered ring.
  • R 1 and R 2 may each be independently selected from H, halogen, preferably iodine or bromine, and G-6 alkyl, preferably C1-3 alkyl, even more preferably methyl. More preferably, R 1 is H and R 2 is selected from halogen, preferably iodine or bromine, and G-6 alkyl, preferably G-3 alkyl, even more preferably methyl. Even more preferably, R 1 is H and R 2 is H or is in the para position and selected from iodine, bromine and methyl.
  • Y may be a linear or branched, optionally substituted, G-C12 hydrocarbyl, more preferably a linear or branched, optionally substituted, G-Go hydrocarbyl, even more preferably a linear or branched, optionally substituted, G-G hydrocarbyl, even more preferably a a linear or branched, optionally substituted, G-G hydrocarbyl.
  • Y may be -(CH 2 )3-.
  • X may be O.
  • albumin binding entity according to Formula (2) may preferably comprise or consist of any one of Formulae (2a)-(2c):
  • the albumin binding entity is conjugated (i.e. covalently linked or attached to) to the -CH- "branching point" via a "spacer".
  • spacer is used herein to specifically refer to the group connecting and spanning the distance between the albumin binding entity and the -CH- "branching point", and/or “spacing" these groups apart from the remaining groups/entities of the conjugate.
  • the spacer may preferably avoid sterical hindrance between the albumin bindi ng entity and the other groups or entities of the inventive conjugate and ensure sufficient mobility and flexibi lity. Futher, the spacer may preferably be designed so as to confer, support and/or allow sufficient HSA binding, high affinity PSMA binding, and rapid and optional ly selective penetration of PSMA positive cells through internalization of the PSMA-conjugate complex.
  • the spacer should preferably comprise at least one C-N bond.
  • Suitable spacers should preferably be stable in vivo. Spacer design may typically depend on the overall conjugate and may preferably be chosen to promote the functionality of the remaining conjugate (e.g. PSMA binding, HSA binding, internalization etc.). Accordingly, spacers may be for instance be rigid or flexible, influencing either lipophilicity or hydrophilicity of the overall conjugate, and so on.
  • the spacer may comprise a li near or branched, optionally substituted G- C2 0 hydrocarbyl comprising up to 5 heteroatoms, more preferably G -C12 hydrocarbyl, even more preferably C 2 -C 6 hydrocarbyl, even more C2-C4 hydrocarbyl.
  • the hydrocarbyl may preferably comprise at least one, optionally up to 4 heteroatoms preferably selected from N.
  • the spacer may be -[CH 10 ] U -N 11 -, wherein R 10 and R 11 may each be independently selected from H and branched, unbranched or cyclic C1-G2 hydrocarbyl and wherein u may be an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0. More preferably, R 10 and R 11 may be H, and u may be an integer selected from 2, 3 or 4. Most preferably, R 10 and R 1 ' may be H and u may be 4.
  • inventive conjugates may preferably comprise a spacer of Formula
  • preferred conjugates according to the invention comprise an albumin bi nding entity of Formula (2a)-(2c) connected to the "branching point" via a spacer of Formula (3a).
  • the spacer may comprise at least one amino acid residue.
  • amino acid residue refers to a specific amino acid monomer as a moiety within the spacer.
  • An “amino acid” is any organic molecule comprising both an acidic (typically carboxy (-COOH)) and an amine (-NH 2 ) functional group. One or both of said groups may optionally be derivatized.
  • the amino and the acidic group may be in any position relative to each other, but amino acids typically comprise 2-amino carboxylic acids, 3-amino carboxylic acids, 4- amino carboxylic acids, etc.
  • the amine group may be attached to the 1 st , 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th (etc.) up to t he 20 th carbon atom of the amino acid(s).
  • the amino acid(s) may be (an) alpha-, beta-, gamma-, delta-, epsilon- (etc.) up to an omega-amino acid(s).
  • the acidic group is a carboxy (-COOH) group.
  • other acidic groups selected from -OPO 3 H, -PO 3 H, -OSO 3 H or-SOsH are also conceivable.
  • the amino acid residue(s) is/are derived from naturally occurring amino acid(s), or derivatives thereof. It is further preferred that the amino acid residues(s) is/are derived from alpha (oc-)amino acid(s), wherein the amino acid(s) may be (a) D- or L-amino acid(s).
  • said amino acid(s) is/are the D- or the L- enantiomer of an amino acid selected from the group arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and/or valine.
  • said amino acid(s) is/are (D-/L-) aspartate, glutamate or lysine.
  • the spacer may comprise 1, 2, 3, 4 or 5 amino acid residue(s), in particular D-aspartate, D- glutamate or L-Lysine residues.
  • the use of the D- enantiomer may provide the beneficial effect of further reducing the rate of metabolisation and thus clearance from the bloodstream.
  • the spacer may comprise between 2 and 3 of such amino acid residues in particular D-aspartate or D-glutamate residues.
  • the spacer may comprise a peptide, which preferably consists of 2 to 5 amino acids, more preferably of 2 to 3 amino acids.
  • the spacer may comprise between 1 and 2 amino acids selected from L-Lysine.
  • inventive conjugates may comprise a spacer of Formula (3b):
  • n 1 or 2
  • n is an integer selected from 1 , 2, 3, 4 or 5, preferably from 2 or 3.
  • the spacer may comprise an amino acid residue connected to th "branching point" via a linear or branched, optional ly substituted, G-C20 hydrocarbyl grou comprising at least one N heteroatom.
  • inventive conjugates may comprise a spacer of Formula (3c):
  • o is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0.
  • o may be 5.
  • the inventive conjugates further comprise a chelator.
  • the terms as imagechelator” or placingchelating moiety are used interchangeably herein to refer to polydentate (multiple bonded) ligands capable of forming two or more separate coordi nate bonds with (coordinating") a central (metal) ion. Specifically, such molecules or molecules sharing one electron pair may also be referred to as contextLewis bases".
  • the central (metal) ion is usually coordinated by two or more electron pairs to the chelating agent.
  • the terms, placedbidentate chelating agent”, touchtridentate chelating agent”, and conjunctiontetradentate chelating agent” are art-recognized and refer to chelating agents having, respectively, two, three, and four electron pairs readily available for simultaneous donation to a metal ion coordinated by the chelating agent.
  • the electron pairs of a chelating agent forms coordinate bonds with a single central (metal) ion; however, in certain examples, a chelating agent may form coordinate bonds with more than one metal ion, with a variety of binding modes being possible.
  • coordinating and coordination refer to an interaction in which one multi-electron pair donor coordinatively bonds (is coordinated") to, i.e. shares two or more unshared pairs of electrons with, one central (metal) ion.
  • the chelating agent is preferably chosen based on its ability to coordinate the desired central (metal) ion, usually a radionuclide as specified herein.
  • the chelator D may be characterized by one of the fol lowing Formulas (4a)-(4jj):
  • the chelator may be DOTA (1 ,4,7, 1 0-tetraazacyclododecane-1 ,4,7, 1 0- tetraacetic acid, which may be characterized by Formula (4a)), NODAGA (2 -(4,7- bis(carboxymethyl)-1 ,4,7-triazonan-1 -yl)-pentanedioic acid, which may be characterized by Formula (4c)), or derivatives thereof.
  • DOTA effectively forms complexes with diagnostic (e.g. 68 Ga) and therapeutic (e.g. 90 Y or l 77 Lu) radionuclides and thus enables the use of the same conjugate for both imaging and therapeutic purposes, i .e.
  • DOTA derivatives capable of complexing Scandium radionuclides 43 Sc, 44 Sc, 47 Sc), including D03AP (which may be characterized by Formula (4hh)), D03AP PrA (which may be characterized by Formula (4ii)), or D03AP ABn (which may be characterized by Formula (4jj)) may also be preferred and are described in Kerdjoudj et al. Dalton Trans., 201 6, 45, 1 398- 1409.
  • chelators in the context of the present invention include N ,N" -bis[2- hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7- triazacyclo-nonane-1 ,4,7-triacetic acid (NOTA), 2-(4,7, 1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetra-azacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydo-nonane-1 -[methyl(2-carboxyethyl)-phosphinic acid]-4,7-bis-[methyl(2-hydroxymethyl)-phosphi nic acid] (NOPO),3,6,9, 1 5-tetra- azabicyclo[9,3, 1
  • the chelator group for example, the DOTA group may be complexed with a central (metal) ion, in particular a radionuclide as defined herein.
  • the chelator group for example DOTA, may not be complexed with a central (metal) ion, in particular a radionuclide as defined herein, and may thus be present in uncomplexed form.
  • the carboxylic acid groups of the chelator can be in the form of a free acid, or in the form of a salt.
  • the inventive conjugates comprise a PSMA binding entity (also referred to as "PSMA binding moiety") herein, which is preferably capable of selectively binding to human PSMA.
  • PSMA binding entity also referred to as "PSMA binding moiety”
  • selectively binding is defined above.
  • the present invention provides compounds according to General
  • Pbm is a PSMA binding entity
  • D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2-(4,7, 1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,
  • X is O, N, S or P
  • R 6 and R 7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl, the spacer comprises at least one C-N bond,
  • the linker is characterized by General Formula (6) as defined herein, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0, or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof.
  • the PSMA binding entity may bind reversibly or irreversibly to PSMA, typical ly with a binding affinity less than about 1 00 ⁇ (micromolar).
  • Human Prostate-specific membrane antigen (also referred to as glutamate carboxypeptidase II (GCPII), folate hydrolase 1 , folypoly-gamma-glutamate carboxypeptidase (FGCP), and N-acetylated-alpha-linked acidic dipeptidase I (NAALADase I)) is a type II transmembrane zinc metal lopeptidase that is most highly expressed in the nervous system, prostate, kidney, and small intestine. It is considered a tumor marker in prostate cancer.
  • the term "Human Prostate-specific membrane antigen" or "PSMA” as used herein preferably refers to the protein encoded by the human FOLH1 gene.
  • the term refers to the protein as characterized under UniProt Acc. No. Q04609 (entry version 1 86, last modified May 1 0, 201 7, or functional variants, isoforms, fragments or (post-translational ly or othweise modified) derivatives thereof.
  • the PSMA-binding entity may generally be a binding entity capable of selectively (and optionally irreversibly) binding to (human) Prostate-Specific Membrane Antigen (cf. Chang Rev Urol. 2004; 6(Suppl 1 0): S1 3-S1 8).
  • the PSMA binding entity is preferably chosen by its abi lity to confer selective affinity towards PSMA.
  • Preferred PSMA binding moieties are described in WO 201 3/022797 A1 , WO 201 5/05531 8 A1 and EP 2862857 A1 , which are incorporated by reference in their entirety herein.
  • the PSMA binding entity may preferably be characterized by General Formula (5):
  • X is selected from O, N, S or P,
  • R 3 , R 4 and R 5 are each independently selected from -COH, -C0 2 H, -SO2H, -SO3H, - SO 4 H, -P0 2 H, -PO 3 H, -P0 4 H 2/ -C(O)-(Ci-Ci 0 )alkyl, -C(O)-O(Ci-C 10 )alkyI, -C(0)-NHR 8 , or - C(0)-NR 8 R 9 ' wherein R 8 and R 9 are each independently selected from H, bond, (C1- C10)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(G- Cio)alkylene, -(CH 2 ) P -NH, -(CH 2 ) P -(C,-Cio)alkyene, -(CH 2 )
  • b, p, q, r, t is each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • b may be an integer selected from 1, 2, 3, 4 or 5
  • R 3 , R 4 and R 5 may each be C0 2 H
  • X may be O.
  • the PSMA binding entity is attached/connected to the - CH- "branching point" via a suitable linker.
  • linker is used herein to specifically refer to the group connecting or linking and thus spanning the distance between the PSMA binding entity and the -CH- "branching point", and/or stiffspacing" the PSMA binding entity apart from the remaining conjugate.
  • the linker may preferably avoid sterical hindrance between the PSMA binding entity and the other groups or entities of the inventive conjugate and ensure sufficient mobi lity and flexibility. Futher, the linker may preferably be designed so as to confer, support and/or allow sufficient HSA binding, high affinity PSMA binding, and rapid and optionally selective penetration of PSMA positive cells through internalization of the PSMA-conjugate complex.
  • PSMA binding entities and in particular preferred PSMA binding entities of General Formula (5), may preferably be linked to the inventive conjugate via a suitable linker as described, e.g. in EP 2 862 857 A1 .
  • Said linker may preferably confer optimized lipophi lic properties to the inventive conjugate to increase PSMA binding and cellular uptake and internalization.
  • the linker may preferably comprise at least one cyclic group and at least one aromatic group (in particular in group Q and W).
  • preferred linkers may be characterized by General Formula (6):
  • X is each i ndependently selected from O, N, S or P,
  • Q is selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl, preferably from substituted or unsubstituted C5-G4 aryl, C 5 -Ci 4 alkylaryl or C5-C14 cycloalkyl
  • W is selected from -(CH 2 ) c -aryl or -(CH 2 ) c -heteroaryl, wherein c is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 .
  • Q is a substituted aryl, alkylaryl or cycloalkyl
  • substituents include, without limitation, halogens (i.e., F, CI, Br, and I); hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; im
  • W may be selected from -(CH 2 )c-napthtyl,-(CH2) c -phenyl, -(CH 2 ) C - biphenyl, -(CH 2 ) c -indolyl, -(CH 2 ) c -benzothiazolyl, wherein c is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. More preferably, W may be selected from -(CH 2 )-napthtyl, -(CH 2 )- phenyl, -(CH 2 )-biphenyl, -(CH 2 )-indolyl or -(CH 2 )-benzothiazolyl.
  • each X may be O.
  • a particularly preferred linker connecting the PSMA binding entity to the inventive conjugate may be characterized by the fol lowing Structural Formula (6a):
  • D may preferably be selected from 1 ,4,7, 10-tetraazacyclododecane-1 ,4,7, 1 0- tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine- N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2-(4,7- bis(carboxymethyl)-1 ,4,7-triazonan-1 -yl)pentanedioic acid (NODAGA), 2-(4,7, 1 0- tris(carboxymethyl)-1 ,4,7,1 0-tetraazacyclododecan-1 -yl)-pentanedi
  • D may preferably be selected from 1 ,4,7, 10-tetraazacyclododecane-1 ,4,7, 1 0- tetraacetic acid (DOTA
  • X may preferably be each independently selected from O, N, S or P. More preferably, each X may be O.
  • R 1 may be H and R 2 may be selected from halogen, preferably iodine or bromine, and C1-6 alkyl, preferably C1-3 alkyl, even more preferably methyl. Even more preferably, R 1 may be H and R 2 may be H or may be in the para position and selected from iodine, bromine and methyl.
  • Y may preferably be selected from a single bond or a linear, branched or cyclic G-
  • Y may be may be a linear or branched, optionally substituted, G -G 2 hydrocarbyl, more preferably a linear or branched, optionally substituted, G-G 0 hydrocarbyl, even more preferably a linear or branched, optionally substituted, G-Q, hydrocarbyl, even more preferably a a linear or branched, optionally substituted, G-C 3 hydrocarbyl.
  • Y may be -(CH 2 ) 3 -.
  • R 3 , R 4 and R 5 may preferably each be i ndependently selected from -COH, -CO2H, - SO 2 H, -SO 3 H, -SO 4 H, -PO 2 H, -PO 3 H, -PO 4 H 2 , -C(0)-(G -Go)alkyl, -C(0)-0(G-Go)alkyl, - C(0)-NHR 8 , or -C(0)-NR 8 R 9 * wherein R 8 and R 9 are each independently selected from H, bond, (C1 -C1 0)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(O)- (G-Go)alkylene, -(CH 2 ) P -NH, -(CH 2 ) P -(G -G 0 )alkyene, -(CH 2 )
  • the spacer may preferably comprise at least one C-N bond. More preferably, the spacer may be characterized by Formula (3a), (3b) or (3c) as defined herein.
  • the linker may preferably be characterized by General Formula (6) as defined herein. More preferably, the linker may be characterized by Formula (6a) as defined herein.
  • Q may preferably be selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl, preferably from substituted or unsubstituted C5-G 4 aryl, C5-C14 alkylaryl or C5-C14 cycloalkyl.
  • W may preferably be selected from -(CH 2 ) c -aryl or -(CH 2 ) c -heteroaryl, wherein c is preferably an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 .
  • A may preferably be an amino acid residue. More preferably, A may be selected from (D-) Aspartate, (D-)Glutamate or (L-Lysine). V may preferably be selected from a single bond, N, or an optionally substituted G-
  • Ci 2 hydrocarbyl comprising up to 3 heteroatoms, wherein said heteroatom is preferably selected from N.
  • n may preferably an integer selected from 1 , 2, 3, 4 or 5, preferably from 1 , 2 or 3
  • m may preferably be 0 or 1 .
  • a, b, p, q, r, t may preferably each be independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0.
  • preferred conjugates according to the present invention may be characterized by General Formula (1 a): d a) wherein
  • D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2 -(4,7,1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1
  • DTPA Diethylenetriaminepentaacetic acid
  • X is each independently selected from O, N, S or P,
  • R 6 and R 7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl
  • R 3 , R 4 and R 5 are each independently selected from -COH, -C0 2 H, -S0 2 H, - SOsH, -SO 4 H, -PO 2 H, -PO 3 H, -P0 4 H 2 , -C(0)-(G-Go)all ⁇ yl, -C(0)-0(G-Go)alkyl - C(0)-NHR 8 , or -C(0)-NR 8 R 9 ' wherein R 8 and R 9 are each independently selected from H, bond, (G-Go)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(G-Go)alkylene, -(CH 2 ) P -NH, -(CH 2 ) p -(G-Go)alkyene, -(CH 2 ) p -NH-C(0)-(CH 2 ) q
  • the spacer comprises at least one C-N bond
  • the linker is characterized by General Formula (6) as defined above, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0, or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof. More preferably, the inventive conjugates may be characterized by General Formula (1 b)
  • D is a chelator, preferably selected from 1 ,4,7,10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)benzyl]- ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane-1 ,4, 7- triacetic acid (NOTA), 2-(4,7-bis( carboxymethyl)-1 ,4,7-triazonan-1 -yl)pentanedioic acid (NODAGA), 2-(4,7,1 0-tris(carboxymethyl)-1 ,4,7, 1 0-tetraazacyclododecan-1 - yl)pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydon
  • Q is selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl,
  • W is selected from -(CH 2 )d-aryl or -(CH 2 )d-heteroaryl
  • a, b, d, p, q, r, s and t are each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0, and
  • the spacer comprises at least one C-N bond, or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof.
  • any one of the following definitions preferably at least two, more preferably at least three, more preferably at least four, or most preferably all of the following definitions may apply for "D”, “Q”, “W”, “a”, “b”, “R 1 “, “R 2 “, R 3 “, “R 4 “ and/or “R 5 " :
  • D may be selected from any suitable chelator (e.g. as defined herein), more preferably D may be selected from from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof. Most preferably, D may be selected from DOTA, NODAGA, D03AP, D03AP PrA or D03AP ABn .
  • Q may be selected from substituted or unsubstituted C5-C7 cycloalkyl.
  • W may be selected from -(CH 2 )-napthtyl, -(CH 2 )-phenyl, -(CH 2 )-biphenyl, -(CH 2 )-indolyl or -(CH 2 )- benzothiazolyl, more preferably W may be -(CH 2 )-napthtyl.
  • a, b may each independently be an integer selected from 0, 1 , 2, 3, 4, 5 or 6.
  • R 1 and R 2 may each independently be selected from H, iodine and G-C 3 alkyl, and R 3 , R 4 and R 5 may each be C0 H.
  • Such preferred conjugates may be characterized by General Formula (1 c):
  • D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof. Most preferably, D may be selected from DOTA, NODAGA, D03AP, D03AP PrA or D03AP AB ,
  • a may be an integer selected from 0, 1 , 2, 3, 4, 5 or 6,
  • R 1 and R 2 are each independently selected from H, iodine or G-G alkyl, and the spacer comprises at least one C-N bond, or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof.
  • a may be 0, and
  • the spacer may be wherein R 10 and R 11 may each be independently selected from H and branched, unbranched or cyclic G-G 2 hydrocarbyl and wherein u may be an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0.
  • the spacer is characterized by Formula (3a). Accordingly, such preferred conjugates may be characterized by General Formula (7a):
  • D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof. Most preferably, D may be selected from DOTA, NODAGA, D03AP, D03AP PrA or D03 AP ABn ,
  • R 1 and R 2 may each be independently selected from H, iodine or G-Cj alkyl, or pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof,
  • preferred conjugates according to the invention may be characterized by Formula (7a)(i) or (7a)(ii):
  • Conjugates characterized by Formula (7a)(i) are also referred to as "PSMA-06" herein.
  • Conjugates characterized by Formula (7a)(ii) are also referred to as "PSMA-03" herein.
  • the spacer comprises at least one amino acid residue, preferably selected from (D-/L-) aspartate, glutamate or lysine.
  • the spacer may comprise at least 1 , 2, 3, 4 or up to five 5 amino acids residue(s), preferably independently selected from (D-/L-) aspartate, glutamate or lysine ami no acid residues.
  • Such conjugates may preferably comprise a spacer according to General Formula (3b) or (3c). Accordingly, such preferred conjugates may be characterized by General Formula (7b):
  • D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA,
  • D may be selected from DOTA, NODAGA, D03AP, D03AP PrA or D03AP ABn ,
  • R 1 and R 2 are each independently selected from H, iodine or C1-C3 alkyl
  • A is an amino acid residue preferably selected from (D-)Aspartate, (D-)Glutamate or
  • V is selected from a single bond, N, or an optionally substituted C1 -C12 hydrocarbyl comprising up to 3 heteroatoms, wherein said heteroatom is preferably selected from N,
  • n is an integer selected from 1 , 2, 3, 4 or 5, preferably from 1 , 2 or 3,
  • a is an integer selected from 1 , 2, 3, 4, 5 or 6. or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof.
  • Conjugates characterized by Formula (7b)(i) are also referred to as PSMA-05 herein.
  • Conjugates characterized by Formula (7b)(ii) are also referred to as "PSMA-07" herein.
  • Conjugates characterized by Formula (7b)(i i i) are also referred to as "PSMA-08" herei n.
  • the present i nvention further encompasses pharmaceutically acceptable salts of the conjugates described herei n.
  • compositions are wel l known to the person ski l led in the art.
  • Pharmaceutical ly acceptable salts of the conjugates of the invention can be prepared by conventional procedures, such as by reacti ng any free base and/or acid of a conjugate accordi ng to the i nvention with at least a stoichiometric amount of the desi red salt- formi ng acid or base, respectively.
  • Pharmaceutically acceptable salts of the i nventive i n include salts with i norganic cations such as sodium, potassium, calcium, magnesium, zi nc, and ammonium, and salts with organic bases.
  • Suitable organic bases i n include N-methyl-D-glucami ne, argmme, benzathi ne, diolamine, olamine, procaine and tromethamine.
  • Pharmaceutically acceptable salts according to the invention also include salts derived from organic or inorganic acids.
  • Suitable anions include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, maleate, mesylate, methylbromide, methylsulfate, napsylate, nitrate, oleate, pamoate, phosphate, polygalacturonate, stearate, succinate, sulfate, subsalicylate, tannate, tartrate, terephthalate, tosylate and triethiodide.
  • the present invention further encompasses the conjugates described herein, wherein the chelating agent D may be complexed with a metal ion (such as a radionuclide) or may not be complexed.
  • a metal ion such as a radionuclide
  • radionuclide refers to isotopes of natural or artificial origin with an unstable neutron to proton ratio that disintegrates with the emission of corpuscular (i.e. protons (alpha-radiation) or electrons (beta-radiation) or electromagnetic radiation (gamma-radiation).
  • corpuscular i.e. protons (alpha-radiation) or electrons (beta-radiation) or electromagnetic radiation (gamma-radiation).
  • chelating agent D may be complexed with any knwon radionuclide.
  • Said radionucl ide which may preferably be useful for cancer imaging or therapy.
  • radionuclides include, without limitation, 94 Tc, 99m Tc, 90 ln, 1 l 1 ln, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 12 Pb, 2 7 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd, or 166 Dy.
  • the choice of suitable radionuclides may depend inter alia on the chemical structure and chelating capability of the chelating agent D, and the intended application of the resulting (complexed) conjugate (e.g.
  • the beta-emitters such as 90 Y, 131 l, 161 Tb and 177 Lu may be used for concurrent systemic radionuclide therapy.
  • Providi ng DOTA as a chelator may advantageously enable the use of either 68 Ga, 43, 4 ' 7 Sc, 177 Lu, 16l Tb, 225 Ac, 213 Bi, 212 Bi, 212 Pb as radionuclides.
  • the present invention further encompasses the inventive conjugates in their esterified form, in particular where free carboxylic acid groups are esterified.
  • esterified compounds may be produg forms of the inventive conjugates.
  • Suitable ester prodrugs include various alkyl esters, including saturated and unsaturated Cs-Cie fatty acids.
  • the conjugates disclosed herein may exist in particular geometric or stereoisomeric forms.
  • compounds may also be optically active.
  • inventive conjugates may also include cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group.
  • a particular enantiomer of a group or conjugate it may be prepared by asymmetric synthesis, or by derivation with a chiral auxi liary, where the resulting diastereomeric mixture is separated and the auxi liary group cleaved to provide the pure desired enantiomers.
  • the group or conjugate contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl
  • diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • a stereoisomer is one stereoisomer of a compound that is substantially free of other stereoisomers of that compound.
  • a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound.
  • a stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound.
  • a typical stereomerically pure compound comprises greater than about 80% by weight of one stereo isomer of the compound and less than about 20% by weight of other stereo isomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 1 0% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereo isomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
  • the present invention relates to the use of the inventive conjugate for the preparation of radiolabeled complexes.
  • radiolabeled complexes preferably comprise a conjugate according to the present invention, and a radionuclide.
  • the chelating agent D preferably coordinates the radionuclide, forming a radiolabeled complex.
  • Suitable radionuclides may be selected from theragnostic metal isotopes and comprise without limitation, 94 Tc, 99m Tc, 90 ln, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 3 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 21 Pb, 2 7 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd, or 166 Dy.
  • the present invention further provides a complex comprising a radionuclide (preferably selected from the group above) and a conjugate according to the invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the inventive conjugate (including pharmaceutically acceptable salts, esters, solavtes or radiolabeled complexes as described herein), and a pharmaceutically acceptable carrier and/or excipient.
  • pharmaceutically acceptable refers to a compound or agent that is compatible with the inventive conjugate and does not interfere with and/or substantially reduce its diagnostic or therapeutic activities.
  • Pharmaceutically acceptable carriers preferably have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject to be treated.
  • Pharmaceutical ly acceptable excipients can exhibit different functional roles and include, without limitation, di luents, fi llers, bulking agents, carriers, disintegrants, binders, lubricants, gl idants, coati ngs, solvents and co-solvents, bufferi ng agents, preservatives, adjuvants, anti-oxidants, wetti ng agents, anti-foaming agents, thickening agents, sweetening agents, flavouri ng agents and humectants.
  • Suitable pharmaceutically acceptable excipients are typical ly chosen based on the formulation of the (pharmaceutical) composition.
  • useful pharmaceutically acceptable excipients in general include solvents, di luents or carriers such as (pyrogen-free) water, (isotonic) saline solutions such phosphate or citrate buffered sal i ne, fixed oi ls, vegetable oi ls, such as, for example, groundnut oi l, cottonseed oi l, sesame oi l, ol ive oi l, corn oi l, ethanol, polyols (for example, glycerol, propylene glycol, polyetheylene glycol, and the l ike); lecithin; surfactants; preservatives such as benzyl alcohol, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the l i ke; isotonic agents such as sugars, polyalcohols such as manitol,
  • pH can be adjusted with acids or bases, such as hydroch loric acid or sodium hydroxide.
  • Buffers may be hypertonic, isotonic or hypotonic with reference to the specific reference medi um, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherei n preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cel ls due to osmosis or other concentration effects.
  • Reference media are e.g. l iquids occurri ng i n in vivo methods, such as blood, lymph, cytosolic l iquids, or other body liquids, or e.g. l iqu ids, which may be used as reference media i n in vitro methods, such as common buffers or l iquids. Such common buffers or l iquids are known to a ski l led person.
  • Liquid (pharmaceutical) compositions admi nistered via injection and i n particular via i .v. i njection should preferably be steri le and stable under the conditions of manufacture and storage.
  • Such compositions are typical ly formulated as parenteral ly acceptable aqueous solutions that are pyrogen-free, have suitable pH, are isotonic and maintai n stabi l ity of the active ingredient(s).
  • suitable pharmaceutical ly acceptable excipients and carriers i n include water, typical ly pyrogen-free water; isotonic sal ine or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions.
  • water or preferably a buffer may be used, which may contain a sodium salt, e.g. at least 50 mM of a sodium salt, a calcium salt, e.g. at least 0,01 mM of a calcium salt, and optional ly a potassium salt, e.g. at least 3 mM of a potassium salt.
  • a sodium salt e.g. at least 50 mM of a sodium salt
  • a calcium salt e.g. at least 0,01 mM of a calcium salt
  • optionally a potassium salt e.g. at least 3 mM of a potassium salt.
  • the sodium, calcium and, optional ly, potassium salts may occur i n the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.
  • examples of sodium salts i n include e.g. NaCI, Nal, NaBr, Na 2 C03, NaHCC , a 2 S04
  • examples of the optional potassium salts i n include e.g. KG, Kl, KBr, K 2 CO 3 , KHCOs, K 2 SO4, and examples of calcium salts i nclude e.g. CaC , Cab, CaBr 2 , CaCCh, CaS0 4 , Ca(OH) 2 .
  • organic anions of the aforementioned cations may be contained i n the buffer.
  • Buffers suitable for i njection purposes as defined above may contai n salts selected from sodium chloride (NaCI), calcium chloride (CaCI 2 ) and optionally potassium chloride (KG), wherei n further anions may be present additional to the chlorides. CaCI 2 can also be replaced by another salt like KG.
  • the salts i n the injection buffer are present i n a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium ch loride (KG) and at least 0,01 mM calcium chloride (CaCI 2 ).
  • the i njection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i .e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherei n preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
  • suitable pharmaceutical ly acceptable excipients and carriers i n include bi nders such as microcrystal l i ne cel lulose, gum tragacanth or gelatin; starch or lactose; sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cel lulose and its derivatives, such as, for example, sodium carboxymethylcel lulose, ethylcel lulose, cel lulose acetate; disintegrants such as algi nic acid; lubricants such as magnesium stearate; gl idants such as stearic acid, magnesium stearate; calcium sulphate, colloidal silicon dioxide and the like; sweetening agents such as sucrose or saccharin; and/or flavoring agents such as peppermint, methyl salicylate, or orange flavor
  • compositions for topical administration can be formulated as creams, ointments, gels, pastes or powders.
  • (Pharmaceutical) compositions for oral administration can be formulated as tablets, capsules, liquids, powders or in a sustained release format.
  • the inventive (pharmaceutical) composition is administered parenterally, in particular via intravenous or intratumoral injection, and is accordingly formulated in liquid or lyophi lized form for parenteral administration as discussed elsewhere herein.
  • Parenteral formulations are typically stored in vials, IV bags, ampoules, cartridges, or pref i I led syringes and can be administered as injections, inhalants, or aerosols, with injections being preferred.
  • the (pharmaceutical) composition may be provided in lyophilized form. Lyophi lized
  • compositions are preferably reconstituted in a suitable buffer, advantageously based on an aqueous carrier, prior to administration.
  • the (pharmaceutical) composition preferably comprises a safe and effective amount of the inventive conjugate(s) or radiolabeled complexe(s).
  • ком ⁇ онент means an amount of the agent(s) that is sufficient to allow for diagnosis and/or significantly induce a positive modification of the disease to be treated. At the same time, however, a meteorsafe and effective amount" is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk.
  • a meteorsafe and effective amount will furthermore vary in connection with the particular condition to be diagnosed or treated and also with the age and physical condition of the patient to be treated, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutical ly acceptable excipient or carrier used, and similar factors.
  • the inventive conjugates are also provided for use in the preparation of a medicament, preferably for treating cancer, in particular for treating and/or preventing prostate cancer, pancreatic cancer, renal cancer or bladder cancer. Kit
  • the present invention relates to a kit comprising the inventive conjugate(s) (including pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof) and/or a pharmaceutical composition(s) of the invention.
  • the kit may comprise at least one further agent as defi ned herein in the context of the pharmaceutical composition, including radionuclides, antimicrobial agents, solubilizing agents or the like.
  • the kit may be a kit of two or more parts comprising any of the components exemplified above in suitable containers.
  • each container may be in the form of vials, bottles, squeeze bottles, jars, sealed sleeves, envelopes or pouches, tubes or blister packages or any other suitable form, provided the container preferably prevents premature mixing of components.
  • Each of the different components may be provided separately, or some of the different components may be provided together (i .e. in the same container).
  • a container may also be a compartment or a chamber within a vial, a tube, a jar, or an envelope, or a sleeve, or a blister package or a bottle, provided that the contents of one compartment are not able to associate physical ly with the contents of another compartment prior to their deliberate mixi ng by a pharmacist or physician.
  • kit or kit-of-parts may furthermore contain technical i nstructions with information on the administration and dosage of any of its components.
  • the present invention relates to the inventive conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical composition or kit for use in medicine and/or diagnostics.
  • inventive conjugates, pharmaceutical compositions or kits are used for human medical purposes.
  • the invention further encompasses these inventive conjugates, pharmaceutical composition or kit for use as a medicament.
  • the inventive conjugates are preferably capable of targeting prostate-specific membrane antigen (PSMA) in a selective manner.
  • the invention thus provides the inventive conjugates, pharmaceutical compositions or kits for use in a method of detecting the presence of cells and/or tissues expressing prostate-specific membrane antigen (PSMA).
  • PSMA is in particular expressed on malignant cancer cells.
  • the term PSMA is in particular expressed on malignant cancer cells.
  • the term refers to a neoplasm characterized by the uncontrolled and usually rapid proliferation of cells that tend to invade surrounding tissue and to metastasize to distant body sites.
  • the term encompasses benign and malignant neoplasms. Malignancy in cancers is typically characterized by anaplasia, invasiveness, and metastasis; whereas benign malignancies typically have none of those properties.
  • the terms include neoplasms characterized by tumor growth as well as cancers of blood and lymphatic system.
  • PSMA may be expressed, optionally in increased amounts, in prostate cancer cells, pancreatic cancer cells, renal cancer cells or bladder cancer cells.
  • the invention provides the inventive conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical composition or kit for use in a method of diagnosing, treating and/or preventing prostate cancer, pancreatic cancer, renal cancer or bladder cancer.
  • the term corposis refers to act of identifying a disease from its signs and symptoms and/or as in the present case the analysis of biological markers (such as genes or proteins) indicative of the disease.
  • the term centretreatment or ⁇ treating" of a disease includes preventing or protecting against the disease (that is, causing the clinical symptoms not to develop); inhibiting the disease (i.e., arresting or suppressing the development of clinical symptoms; and/or relieving the disease (i.e., causing the regression of clinical symptoms).
  • the termiserprophylaxis will be understood to constitute a type ofußtreatment” that encompasses both bakingpreventing" and suppressing.”
  • the term compassion thus includes consultprophylaxis".
  • the term drawnsubject generally includes humans and non-human animals and preferably mammals (e.g., non-human primates, including marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, and baboons, macaques, chimpanzees, orangutans, gorillas; cows; horses; sheep; pigs; chicken; cats; dogs; mice; rat; rabbits; guinea pigs etc.), including chimeric and transgenic animals and disease models.
  • the term appearingsubject preferably refers a non-human primate or a human, most preferably a human.
  • the uses and methods described herein and relating to the diagnosis, treatment or prophylaxis of cancer, in particular prostate cancer, pancreatic cancer, renal cancer or bladder cancer may preferably comprise the steps of (a) administering the inventive conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical composition or kit to a patient, and (b) obtaining a radiographic image from said patient.
  • inventive conjugate including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof
  • pharmaceutical composition or kit may preferably comprise the steps of (a) administering the inventive conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical composition or kit to a patient, and (b) obtaining a radiographic image from said patient.
  • inventive conjugates, pharmaceutical compositions or kits are typical ly administered parenterally.
  • Administration may preferably be accompl ished systemically, for instance by intravenous (i.v.), subcutaneous, intramuscular or intradermal injection.
  • administration may be accomplished locally, for instance by intra-tumoral injection.
  • inventive conjugates, pharmaceutical compositions or kits may be administered to a subject in need thereof several times a day, dai ly, every other day, sweekly, or monthly.
  • treatment, diagnosis or prophylaxis is effected with an effective dose of the inventive conjugates, pharmaceutical compositions or kits.
  • Effective doses of the inventive conjugates may be determined by routine experiments, e.g. by using animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models.
  • Therapeutic efficacy and toxicity of inventive conjugates or radiolabeled complexes can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • the data obtained from the cell culture assays and animal studies can be used in determining a dose range for use in humans.
  • the dose of said conjugates lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • therapeutical ly or diagnostically effective doses of the inventive conjugates may range from about 0.001 mg to 1 0 mg, preferably from about 0.01 mg to 5 mg, more preferably from about 0.1 mg to 2 mg per dosage unit or from about 0.01 nmol to 1 mmol per dosage unit, in particular from 1 nmol to 1 mmol per dosage unit, preferably from 1 micromol to 1 mmol per dosage unit. It is also envisaged that therapeutical ly or diagnostically effective doses of the inventive conjugates may range (per kg body weight) from about 0.01 mg/kg to 1 0 g/kg, preferably from about 0.05 mg/kg to 5 g/kg, more preferably from about 0.1 mg/kg to 2.5 g/kg.
  • the inventive conjugates may preferably be administered at lower doses than other PSMA ligands.
  • the inventive conjugates particularly lend themselves for theragnostic applications involving the targeting of PSMA-expressing cells.
  • the term closertherangostic includes consulttherapeutic-only", whildiagnostic-only” and practicethepeutic and diagnostic” applications.
  • the present invention relates to an in vitro method of detecting the presence of cells and/or tissues expressing prostate-specific membrane antigen (PSMA) comprising (a) contacting said PSMA-expressing cells and/or tissues with the inventive conjugates (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical compositions or kits and (b) applying detection means, optionally radiographic imaging, to detect said cells and/or tissues.
  • PSMA prostate-specific membrane antigen
  • radiographic imaging may be accomplished using any means and methods known in the art.
  • radiographic imaging may involve positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • the targeted cells or tissues detected by radiographic imaging of the inventive conjugate may preferably comprise (optionally cancerous) prostate cells or tissues, (optionally cancerous) spleen cel ls or tissues, or (optionally cancerous) kidney cells or tissues.
  • the presence of PSMA-expressing cells or tissues may be indicative of a prostate tumor (cell), a metastasized prostate tumor (cel l), a renal tumor (cell), a pancreatic tumor (cell), a bladder tumor (cell), and combinations thereof.
  • inventive conjugates including pharmaceutical ly acceptable salts, esters, solvates and radiolabeled complexes thereof
  • pharmaceutical compositions and kit may particularly be employed for diagnosis (and optional ly treatment) of prostate cancer, renal cancer, pracreatic cancer, or bladder cancer.
  • FIGURE 1 Chromatograms of the HPLC-based quality control of (A) 177 Lu-PSMA-ALB-01 , (B) 177 Lu-PSMA-ALB-03, (C) 177 Lu-PSMA-ALB-04, (D) 177 Lu-PSMA-ALB-05, (E) 177 Lu-PSMA-ALB- 06, (F) 177 Lu-PSMA-ALB-07, and (G) 177 Lu-PSMA-ALB-08 labeled at 50 MBq/nmol.
  • A&C Data obtained in PSMApos PC-3 PIP cells.
  • B&D Data obtained in PSMAneg PC-3 flu cells.
  • FIGURE 5 Biodistribution data of PC-3 PIP/flu tumor-bearing mice treated with 177 Lu-PSMA- ALB-01 and 1 7 Lu-PSMA-ALB-03 (A), 177 Lu-PSMA-ALB-04 and 177 Lu-PSMA-ALB-05 (B) and 177 Lu-PSMA-ALB-06, 177 Lu-PSMA-ALB-07 and 177 Lu-PSMA-ALB-08 (C).
  • FIGURE 6 A conclusive selection of all (A) the tumor uptake, (B) the tumor/blood ratio, (C) the tumor/kidney ratio and (D) the tumor/liver ratio of 177 Lu-PSMA-ALB-01 -08.
  • Table 1 Analytical Data of PSMA-ALB-01 /03/04/201706/07/08.
  • the peptidomimetic pharmacophore for PSMA (L-Glu-NH-CO-NH-L-Lys binding entity; step 1 -6) was synthesized analogically as described by Eder et al. . Bioconjug. Chem. 201 2, 23: 688-697.
  • the linker moiety (2-naphthyl-L-Ala-NH-CO-trans-CHX-N3 or 2- naphthyl-L-Ala-NH-CO-trans-CH x -Me-NH 2 ; step 7-1 0) was prepared accordi ng to standard Fmoc (9-fluorenylmethyloxycarbonyl) protocol as previously introduced by Benesova et al.
  • PSMA-ALB-01 For synthesis of PSMA-ALB-01 , time-efficient mecanical head-to-tai l" click coupling of the purified PSMA-precursor with the free azido group and the purified albumin-binding moiety [4-(p-iodophenyl)butyric acid-L-Lys] with propargyl-Gly (step 1 1 -1 7) was employed. After the efficient coupling of these two precursors via a triazole ring (step 1 8), an additional purification was performed to remove an excess of CuSCV5 H 2 0. Final ly, PSMA-ALB-01 was obtained by the conjugation of the DOTA chelator in a form of its active ester (DOTA-NHS ester; step 1 9).
  • DOTA-NHS ester DOTA-NHS ester
  • PSMA-ALB-03 For the preparation of PSMA-ALB-03, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, DOTA tris(tBu)-ester conjugation, Alloc deprotection and 4-(p- iodophenyl)butyric acid conjugation fol lowed (step 1 1 -1 6). Final ly, PSMA-ALB-03 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H 2 0 mixture (step 1 7).
  • PSMA-ALB-04 For the synthesis of PSMA-ALB-04, time-efficient tillhead-to-tail" coupling of the resin- coated PSMA-precursor with the DOTA-conjugated L-Lys and the purified albumin-binding moiety [4-(p-iodophenyl)butyric acid-L-Lys] through direct conjugation of two secondary amines (step 1 1 -1 8) was employed. After the efficient coupling of these two precursors using suberic acid bis(N-hydroxysuccinimide) ester (step 1 9), PSMA-ALB-04 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H 2 0 mixture (step 20).
  • PSMA-ALB-05 For the preparation of PSMA-ALB-05, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, 4-(p-iodophenyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation followed (step 1 1 -1 9). PSMA-ALB-05 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H 2 0 mixture (step 20).
  • PSMA-ALB-06 For the synthesis of PSMA-ALB-06, straight one-way synthesis on the resi n support was employed. After the Fmoc-L-Lys(Alloc)-OH coupl i ng to PSMA-precursor, Fmoc deprotection, DOTA tris(tBu)-ester conjugation, Al loc deprotection and p-(tolyl)butyric acid conjugation followed (step 1 1 -1 6). Finally, PSMA-ALB-06 was obtained by agitation and subsequent cleavage from the resi n with TFA:TI PS:H 2 0 mixture (step 1 7).
  • PSMA-ALB-07 For the preparation of PSMA-ALB-07, straight one-way synthesis on the resi n support was employed. After the Fmoc-L-Lys(Al loc)-OH coupl ing to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, 4-(p-iodophenyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation fol lowed (step 1 1 -22).
  • PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resi n with TFA:TI PS:H 2 0 mixture (step 23).
  • PSMA-ALB-08 straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupl ing to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, p-(tolyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation followed (step 1 1 -22).
  • PSMA-ALB-08 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H 2 0 mixture (step 23).
  • 2-Chlorotrityl chloride resin ⁇ (2-CT-Resin; Merck; Catalog number 85501 70005), 0.30 mmol, substitution capacity 1 .63 mmol/g, 1 00-200 MESH, 1 % DVB, total swel ling volume in CH 2 CI 2 >4.2 mL/g, [1 84 mg] ⁇ in 5mL syringe with the filter and combi stopper was first agitated in anhydrous dichloromethane (DCM) for 45 min.
  • DCM hydrous dichloromethane
  • the 2-CT-resin was then washed three times with anhydrous DCM and followed by reaction with 1 .2 equiv of Alloc (/V-allyloxycarbonyl) as well as Fmoc (N- fluorenylmethoxycarbonyl) protected L-lysine ⁇ (Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.36 mmol, 452.50 g/mol, [1 63 mg], (1 ) ⁇ and 4.8 equiv of N,N- di isopropylethylamine ⁇ (DIPEA), 1 .44 mmol, 129.24 g/mol, 0.742 g/ml, [251 pL] ⁇ in 3 mL of anhydrous DCM.
  • Alloc /V-allyloxycarbonyl
  • Fmoc N- fluorenylmethoxycarbonyl protected L-lysine ⁇ (Fmoc-Lys(
  • the coupling of the first protected ami no acid on the resin (2) proceeded over the course of 1 6 h with the gentle agitation.
  • the L-lysine-immobilized resin (2) was washed three times with DCM1 and three times with DCM2. Unreacted chlorotrityl groups remaining on the resin were washed five times with the mixture of DCM, methanol (MeOH), and DIPEA in a ratio of 1 7:2:1 (20 mL).
  • the resin with Alloc and Fmoc protected L-lysine was washed three times with DCM1 , three times with DCM2, three times with ⁇ , ⁇ -dimethylformamide (DMFI ), and, finally, three times with DMF2.
  • the L-lysine-immobilized resin with one free NH 2 -group (3) was added afterwards in one portion to the solution of the isocyanate of the glutamyl moiety iii and stirred for 1 6 h in order to obtain resin-immobilized bis(tBu)-Glu-urea-Lys(Alloc) (4).
  • the resin was then washed three times with DCM1 , three times with DCM2, three times with DMF1 , and, fi nal ly, three times with DMF2.
  • the resin was additionally washed ten times with 1 % DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral ⁇ (sodium diethyldithiocarbamate trihydrate; Sigma; Catalog number D3506-1 00G), 225.31 g/mol ⁇ in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 ml_ DMF2).
  • the resin-immobilized and bis(tBu)-protected Glu-urea-Lys (5) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , three times with DCM2, and, finally, three times with diethylether (Et 2 0) and dried under vacuum.
  • PSMA binding entity (5) was used for the next reaction in order to synthesize all seven compounds (PSMA-ALB- 01 /03/04/201706/07/08).
  • the resin-immobilized and bis(tBu)-protected binding entity (5) was first agitated anhydrous DCM for 45 min. Pre-swollen pharmacophore was washed three times wi DCM2, three times with DMF1 , and three times with DMF2.
  • the conjugation of the chelator to the resin-immobilized compound (1 1 B) was performed with 2 equiv of DOTA-tris(t-Bu)ester ⁇ ([2 -(4,7, 10-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.20 mmol, 572.73 g/mol [1 1 5 mg] ⁇ .
  • the chelator building block was activated with 1 .98 equiv of HBTU ⁇ (Sigma; Catalog number 12804-25G-F), 0.1 98 mmol, 379.24 g/mol, [75 mg] ⁇ in the presence of 4 equiv of DIPEA ⁇ 0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [70 pL] ⁇ in anhydrous DMF.
  • DIPEA Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 1 B).
  • the coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
  • the resulting compound (12B) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , and, finally, three times with DCM2.
  • the resin was then washed three times with DCM1 , three times with DCM2, three times with DMFl , and, finally, three times with DMF2.
  • the resin was additionally washed ten times with 1 % DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral ⁇ (Sigma; Catalog number D3506-100G), 225.31 g/mol ⁇ in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 mL DMF2).
  • the resulting compound (1 3 B) was then washed three times with DMF1 and three times with DMF2.
  • the final compound PSMA-ALB-03 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H 2 0 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
  • the conjugation of the chelator to the resin-immobilized compound (1 1 B) was performed with 3 equiv of DOTA-tris(f-Bu)ester ⁇ ([2-(4,7,1 0-tris(2-(f-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.30 mmol, 572.73 g/mol [1 71 mg] ⁇ .
  • the chelator building block was activated with 2.97 equiv of HBTU ⁇ (Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg] ⁇ in the presence of 4 equiv of DIPEA ⁇ 0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL] ⁇ in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobi lized and the DMF pre-swollen compound (1 1 B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
  • the resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Dde-protecting group from the resulting compound (12 B) was realized by washing with the mixture of 2 % hydrazine in DMF twice for 5 min and then once again for 1 0 min in order to obtain the product (1 3B).
  • the 2-CT resin was then washed three times with anhydrous DCM and followed by reaction with 1 .2 equiv of Dde as well as Fmoc protected L-lysine ⁇ (Dde-Lys(Fmoc)-OH; Bachem; Catalog number E-3385.0001 ), 0.24 mmol, 532.64 g/mol, [128 mg] (1 5 B) ⁇ and 4.8 equiv of DIPEA ⁇ 0.96 mmol, 129.24 g/mol, 0.742 g/mL, [1 67 pL] ⁇ in 3 mL of anhydrous DCM.
  • the L-lysine-immobilized resin (1 6B) was washed three times with DCM1 and three times with DCM2. Unreacted chlorotrityl groups remaining on the resin were washed five times with the mixture of DCM, MeOH, and DIPEA in a ratio of 1 7:2:1 (20 mL).
  • Dde protected L-lysine was then washed three times with DMF1 and three times with DMF2, three times with DCM1 , three times with DCM2 and, finally, three times with Et 2 0 and dried under vacuum.
  • Such prepared resin-coated Dde protected L-lysine (1 7B) was split into two portions and one of them was used for the next reaction.
  • This resin-coated product was agitated in anhydrous DCM for 45 min and subsequently washed three times with DMF and three times with DMF2.
  • the resin with Dde protected L-lysine and iodophenyl-butyric acid (18B) was washed three times with DMF1 and three times with DMF2.
  • Selective removal of Dde-protecting group from the resulting compound (18B) was realized by washing with the mixture of 2 % hydrazine in DMF twice for 5 min and then once again for 10 min in order to obtain the product (19B).
  • the albumin-targeting moiety (20B) was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of 5% TFA in DCM.
  • the mixture of solvents from the product was evaporated, crude product dissolved in ACN and water in a ratio of 1 :1 and purified via RP-HPLC.
  • the outline of the above described synthesis is summarized in Scheme 5.
  • the resulting compound (21 B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with Et 2 0 and dried under vacuum.
  • the final compound PSMA-ALB-04 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H 2 0 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
  • PSMA precursor (Albumin precursor) (PSMA-ALB-04) a) DMF, DIPEA; b) TFA:TIPS:H 2 0;
  • the resulting compound (12 B) was then washed three times with DMF1 and three times with DMF2.
  • Selective removal of Fmoc-protecting group from the resulting compound (12B) was realized by washing with the 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 (1 3B).
  • the resin was additionally washed ten times with 1 % DIPEA in DMF (300 ⁇ _ DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 mi n with a solution of cupral ⁇ (Sigma; Catalog number D3506-1 00G), 225.31 g/mol ⁇ in DMF2 at the concentration of 1 5 img/mL (450 mg cupral in 30 mL DMF2).
  • the resulting compound (1 7B) was then washed three times with DMF1 and three times with DMF2.
  • the conjugation of the chelator to the resin-immobilized compound (1 7B) was performed with 3 equiv of DOTA-tris(t-Bu)ester ⁇ ([2 -(4,7, 1 0-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.30 mmol, 572.73 g/mol [1 71 mg] ⁇ .
  • the chelator building block was activated with 2.97 equiv of HBTU ⁇ (Sigma; Catalog number 1 2804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg] ⁇ in the presence of 4 equiv of DIPEA ⁇ 0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL] ⁇ in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 7B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
  • Such product (1 8B) washed three times with DMF1 and three times with DMF2, three times with DCM1 , three times with DCM2 and, final ly, three times with Et 2 0 and dried under vacuum.
  • the final compound PSMA-ALB-05 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H 2 0 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in ratio of 1 :1 and purified via RP-HPLC.
  • the conjugation of the chelator to the resin-immobi lized compound (1 1 ) was performed with 2 equiv of DOTA-tris(t-Bu)ester ⁇ ([2 -(4,7, 1 0-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7, 1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.20 mmol, 572.73 g/mol [1 1 5 mg] ⁇ .
  • the chelator building block was activated with 1 .98 equiv of HBTU ⁇ (Sigma; Catalog number 12804-25G-F), 0.1 98 mmol, 379.24 g/mol, [75 mg] ⁇ in the presence of 4 equiv of DIPEA ⁇ 0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL] ⁇ in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 1 ). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
  • the resin was then washed three times with DCM1 , three times with DCM2, three times with DMF1 , and, final ly, three times with DMF2.
  • the resin was additionally washed ten times with 1 % DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral ⁇ (Sigma; Catalog number D3506-100G), 225.31 g/mol ⁇ in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 mL DMF2).
  • the resulting compound (1 3) was then washed three times with DMF1 and three times with DMF2.
  • the resulting compound (1 4) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , three times with DCM2, and, finally, three times with Et20 and dried under vacuum.
  • the final compound PSMA-ALB-06 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H 2 0 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1 :1 and purified via RP-HPLC.
  • the resulting compound (1 2 B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 2 B) was realized by washing with the 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 (1 3B).
  • the resin was additionally washed ten times with 1 % DIPEA in DMF (300 ⁇ _ DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral ⁇ (Sigma; Catalog number D3506-1 00G), 225.31 g/mol ⁇ in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 mL DMF2).
  • the resulting compound (1 9B) was then washed three times with DMF1 and three times with DMF2.
  • the conjugation of the chelator to the resin-immobilized compound (1 9B) was performed with 3 equiv of DOTA-tris(t-Bu)ester ⁇ ([2 -(4,7, 1 0-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.30 mmol, 572.73 g/mol [1 71 mg] ⁇ .
  • the chelator bui lding block was activated with 2.97 equiv of HBTU ⁇ (Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg] ⁇ in the presence of 4 equiv of DIPEA ⁇ 0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL] ⁇ in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 7B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
  • Such product (20B) washed three times with DMF1 and three times with DMF2, three times with DCM1 , three times with DCM2 and, finally, three times with Et 2 0 and dried under vacuum.
  • the final compound PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in ratio of 1:1 and purified via RP-HPLC.
  • the resulting compound (1 2 B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 2 B) was realized by washing with the 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 (1 3 B).
  • the resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2.
  • the resin was additionally washed ten times with 1% DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral ⁇ (Sigma; Catalog number D3506-100G), 225.31 g/mol ⁇ in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2).
  • the resulting compound (19B) was then washed three times with DMF1 and three times with DMF2.
  • the conjugation of the chelator to the resin-immobilized compound (19B) was performed with 3 equiv of DOTA-tris(t-Bu)ester ⁇ ([2 -(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,10-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 137076-54- 1 ), 0.30 mmol, 572.73 g/mol [171 mg] ⁇ .
  • the chelator building block was activated with 2.97 equiv of HBTU ⁇ (Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg] ⁇ in the presence of 4 equiv of DIPEA ⁇ 0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL] ⁇ in anhydrous DMF.
  • DIPEA Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (17B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
  • PSMA-ligands 177 Lu-PSMA-ALB-01 /-03/-04/-05/-06/-07/-08 were synthesized as described above (see Examples 1 and 2).
  • the reference compound (PSMA-61 7) was purchased from Advanced Biochemical Compounds (ABX GmbH, Radeberg, Germany). No-carrier added 177 Lu in 0.05 M HCI was provided by Isotope Technologies Garching (ITG GmbH, Germany).
  • the stock solution of PSMA-61 7 was prepared by di lution in MilliQ water to a final concentration of 1 mM.
  • l 77 Lu-PSMA-ALB-01 /-03/-04/-05/-06/-07/-08 were diluted in Mil liQ water/DMSO to obtain a final concentration of 1 mM.
  • All compounds were labeled with 177 Lu in a 1 :5 mixture of sodium acetate (0.5 M, pH 8) and HCI (0.05 M, pH ⁇ 1 ) at pH 3.5-4.5.
  • the compounds were labeled with 177 Lu at specific activities between 5-50 MBq/nmol, depending on the experimental conditions.
  • the reaction mixture was incubated for 1 5 min at 95°C, followed by a quality control using high-performance liquid chromatography with a C-1 8 reversed-phase column (XterraTM MS, C1 8, 5 ⁇ , 1 50x4.6 mm; Waters).
  • the mobi le phase consisted of MilliQ water containing 0.1 % trifluoracetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% B to 20% A and 80% B over a period of 1 5 min at a flow rate of 1 .0 mL/min.
  • the radioligands were diluted in MilliQ water containing Na-DTPA (50 ⁇ (micromolar)) prior to injection into HPLC. Determination of the n-Octanol/PBS Distribution Coefficient
  • 177 Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 and PSMA-617 were labeled with 177 Lu at a specific activity of 50 MBq/nmol.
  • the radioligand (0.5 MBq; 10 pmol, 25 ⁇ [_) was then added to a reagent tube containing 1475 ⁇ of PBS pH 7.4 and 500 ⁇ _ of n-octanol.
  • the vials were vortexed vigorously followed by a centrifugation step for phase separation.
  • Plasma binding of l77 Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 and l77 Lu-PSMA-617 was determined using an ultrafiltration assay.
  • the compounds were labeled with ' 77 Lu at a specific activity of 50 MBq/nmol and incubated in human plasma samples or PBS at room temperature.
  • the free and plasma-bound fractions were separated using a centrifree ultrafiltration device (4104 centrifugal filter units [Millipore]; 30000 Da nominal molecular weight limit, methylcellulose micropartition membranes).
  • the incubated solution was loaded to the ultrafiltration device and centrifuged at 2500 rpm for 40 min at 20°C. Samples from the filtrate were taken an analyzed for radioactivity in a gamma-counter.
  • the amount of plasma-bound compound was calculated as the fraction of radioactivity measured in the filtrate relative to the corresponding loading solution (set to 100%).
  • Routine cell culture was performed twice a week using PBS/EDTA (2 mM) for washing the cells and trypsin for detachment of the cells.
  • the cells were seeded in 12-well plates ( ⁇ 3 x 1 0 s cel ls in 2 mL RPMI medium/well) allowing adhesion and growth overnight at standard conditions. The supernatant was removed and the cel ls washed with PBS pH 7.4 prior to the addition of RPMI medium without supplements (975 pL/well).
  • the compounds were labeled with 177 Lu at a specific activity of 5 MBq/nmol and diluted to 1 .5 MBq/mL in 0.05% bovine serum albumin (BSA)/0.9% NaCI solution to prevent adherence to plastic vessels.
  • BSA bovine serum albumin
  • the cells were incubated with 25 ⁇ _ (-37.5 kBq)/well radiolabeled PSMA ligands at standard conditions for 2 h and 4 h, respectively. After incubation, the cells were washed three times with ice-cold PBS and the total uptake of the radioligands was determined (PSMA- bound fraction on the surface and internalized fraction). The fraction of internalized radioligand was evaluated in cells washed with ice-cold PBS, followed by a 1 0 min incubation with stripping buffer (0.05 M glycine stripping buffer in 1 00 mM NaCI, pH 2.8) and an additional washing step with ice-cold PBS.
  • stripping buffer 0.05 M glycine stripping buffer in 1 00 mM NaCI, pH 2.8
  • n-Octanol/PBS Distribution Coefficient l 77 Lu-PSMA-ALB-01, -03, -04 and -06 showed similar n-octanol/PBS distribution coefficients (LogD value), while the coefficients of 177 Lu-PSMA-ALB-05, -07 and -08 indicated slightly more hydrophilic compounds.
  • the data showed that the introduction of an albumin-binding entity reduces the hydrophilicity as compared to the reference compound 177 Lu-PSMA-617, however, all compounds are still hydrophilic with logD values > 2.7 (Fig 2).
  • 177 Lu-PSMA-ALB-01 , -03, -04, -05, -06, -07 and -08 were characterized in vivo. Therefore, immunodeficient Balb/c nude mice were inoculated with PSMApos PC-3 PIP and PSMAneg PC-3 flu cells. After intravenous (i.v.) application of the ligands, extensive biodistribution and SPECT/CT studies were performed. Tumor uptake, tumor/blood ratio, tumor/kidney ratio and tumor/liver ratio of 177 Lu-PSMA-ALB-01 -08 are summarized in Figure 6.
  • mice were obtained from Charles River Laboratories, Sulzfeld, Germany, at the age of 5-6 weeks.
  • Female, athymic nude Balb/c mice were subcutaneously inoculated with PC-3 PIP cells (6 x 10 6 cells in 1 00 ⁇ Hank's balanced salt solution (HBSS) with Ca 2+/ Mg 2+ ) on the right shoulder and with PC-3 flu cells (5 x 1 0 6 cells in 1 00 ⁇ HBSS Ca 2+ Mg 2+ ) on the left shoulder. Two weeks later, the tumors reached a size of about 200-300 mm 3 suitable for the performance of the biodistribution and imaging studies.
  • HBSS Hank's balanced salt solution
  • 177 Lu-PSMA-ALB-01 and 177 Lu-PSMA-ALB-03 were investigated over a period of eight days. Compounds 177 Lu-PSMA-ALB-01 and 177 Lu-PSMA- ALB-03 showed highly similar tissue distribution profiles (Fig. 5A).
  • Salivary glands 5.52 + 1 .08 5.20 + 0.73 4.45 + 0.56 3.38 + 0.32 3.96 ⁇ 0.98 2.22 ⁇ 0.38
  • Salivary glands 5.60 + 0.70 5.02 ⁇ 1.17 5.49 + 0.59 4.69 + 0.33 3.45 ⁇ 1.09 2.19 ⁇ 0.1
  • Salivary glands 1.67 ⁇ 0.27 1.55 + 0.06
  • tissue distribution of 177 Lu-PSMA-ALB-04 and 177 Lu-PSMA-ALB-05 was investigated over a period of eight days (Fig. 5B).
  • Salivary glands 9.93 ⁇ 2.33 n/d n/d 12.5 ⁇ 0.42 10.6 ⁇ 0.42 4.08 ⁇ 0.44
  • Salivary glands 0.46 ⁇ 0.09 0.37 ⁇ 0.04
  • the tissue distribution of 177 Lu-PSMA-ALB-06, -07 and -08 was investigated up to three days post injection (Fig.5C).
  • Salivary glands n/d 3.26 ⁇ 0.16 n/d 0.52 + 0.11 n/d 0.24 + 0.03

Abstract

The present invention provides novel compounds that are useful as radiopharmaceuticals, imaging agents and for treatment of cancer.

Description

Novel PSMA-binding agents and uses thereof
The present invention relates to novel compounds and radiolabeled complexes comprising a chelating agent, a PSMA-binding entity and an albumin-binding entity connected via suitable linkers and spacers, which are envisaged for use as diagnostic and/or therapeutic radiopharmaceuticals. Specifically, the compounds and complexes according to the invention lend themselves as (theragnostic) tracers, imaging agents and therapeutic agents for detecting PSMA-expressing target cells and tissues and treating and diagnosing cancer.
Prostate cancer (PCa) is the leading cancer in the US and European population. 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. According to the American Cancer Society, approximately 1 61 ,000 new cases of prostate cancer are diagnosed each year in USA. The 5-year survival rate of patients with stage IV metastatic prostate cancers is only about 29%.
Once a metastatic PCa becomes hormone-refractory there are only a few therapy options left, often with rather poor clinical success. According to the current medical guidelines, antimitotic chemotherapy with docetaxel is typically recommended. However, treatment is often associated with severe side effects, and only marginally improved survival rates. Early diagnosis and close monitoring of potential relapses are therefore crucial. Prostate cancer diagnosis is based on examination of histopathological or cytological specimens from the gland. Existing imaging techniques for therapeutic monitoring of progressing or recurring prostate cancer, include computed tomography (CT), magnetic resonance (MR) imaging and ultrasound, but are often insufficient for effective monitoring and management of the disease. Consequently, there is a high cli nical demand for more effective tools for both early diagnosis and treatment of PCa. It is well known that tumor cells may express unique proteins exhibiting a modified structure due to mutation, or may over-express normal (i.e. non-mutated) proteins that are normally produced in extremely small quantities in non-malignant cells. Tumor antigens may be broadly classified into two categories based on their expression pattern: Tumor-Specific Antigens (TSA), which are present only on tumor cells and not on non-malignant cells and Tumor-Associated Antigens (TAA), which are present on some tumor cells and also non- malignant cells. TSAs typically emerge as a result of the mutation of protooncogenes and tumor suppressors which lead to abnormal protein production, whereas TAA expression is general ly caused by mutation of other genes unrelated to the tumor formation.
The expression of such proteins on the surface of tumor cel ls offers the opportunity to diagnose and characterize disease by detecting such tumor markers. Proteinaceous binding agents or small molecule drugs carrying visualizable labels and specifically recognizing such tumor markers are typically employed for diagnosing and imaging cancers under non- invasive conditions.
A promising new series of low molecular-weight imaging agents targets the prostate- specific membrane antigen (PSMA). PSMA, also known as folate hydrolase I (FOLH1 ), is a trans-membrane, 750 amino acid type II glycoprotein. The PSMA gene is located on the short arm of chromosome 1 1 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 /V-acetyl-aspartyl-glutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (Nan, F.; et al. J Med Chem 2000, 43, 772-774).
PSMA is (i) mainly restricted to the prostate (although is also detected in lower amounts in the neovasculature of numerous other solid tumors, including bladder, pancreas, lung, and kidney cancers, but not in normal vasculature), (ii) abundantly expressed as protein at all stages of prostate cancer (in amounts of up to 1 06 PSMA molecules per cancer cell) (iii) presented at the cell surface but not shed into the circulation, and (iv) associated with enzymatic or signaling activity. Moreover, PSMA expression is further up-regulated in poorly differentiated, androgen-i nsensitive or metastatic cancers and the expression usually correlateds with disease progression. The unique expression of PSMA makes it an important marker of prostate cancer (and a few other cancers as well). Furthermore, PSMA represents a large extracel lular target for imaging agents. PSMA is internalized after ligand binding and, thus, it is not only an excel lent target for targeted radionuclide therapy (using particle-emitting radionuclides) but also for other therapeutic strategies including the tumor cel l-specific delivery of immunotoxins, retargeting of immune cells, pro-drug activation, PSMA vaccines, and plasmid DNA and adenoviral immunizations. Because of low expression levels in healthy tissue, PSMA has additional ly the potential for high-dose therapy, with minimized side effects.
In the past, several PSMA-targeting agents carrying therapeutic or diagnostic moieties were developed. The FDA-approved radio-immunoconjugate of the anti-PSMA monoclonal antibody (mAb) 7E1 1 , known as PROSTASCINT®, has been used to diagnose prostate cancer metastasis and recurrence. The success of this radiopharmaceutical agent is limited due to the fact that this antibody binds to the intracellular domain of PSMA, hence, can target only dead cells. Moreover, the use of monoclonal antibodies and antibody fragments as imaging agents is often limited due to their slow renal clearance, heterogenous distribution, poor tumor penetration and immunogenic potential. In order to overcome these problems, various small-molecule PSMA targeting agents capable of binding to the extracellular domain of PSMA were developed for PET/CT and SPECT/CT imaging, including radiolabeled N-[N-[(S)- 1 ,3-dicarboxypropyl]carbamoyl]-S-[1 1 C]methyl-l-cysteine (DCFBC) and several urea-based peptidomimetic PSMA-inhibitors (cf. Bouchelouche et al. Discov Med. 201 0 Jan; 9(44): 55- 61 ), including MIP-1 095 (H illier et al. Cancer Res. 2009 Sep 1 ;69(1 7):6932-40), a PSMA ligand currently in clinical evaluation, and DOTA-conjugated PSMA-inhibitor PSMA-61 7 developed by Benesova et al (JNM 201 5, 56: 914-920 and EP 2862 857 A1 ), which distributes throughout the body and rapidly clears from the blood 0 Nucl Med. 201 5;56(1 1 ):1 697-705). However, although rapid and systemic access advantageously facilitates tumor targeting and - penetration, currently avai lable PSMA-targeting agents bear the risk of mediating unspecific "off-target" interactions in normal tissues expressing the target, and of accumulation of the radiopharmaceuticals in excretory organs (such as the kidneys). Thereby, non-tumorous tissues may be exposed to radiation doses ultimately leading to irreversible tissue damage. It was demonstrated that different radiolabeled smal l- molecule PSMA-targeting agents (including PSMA-61 7) accumulate in patients' lacrimal and salivary glands and may cause damage to the glandular tissue, especially if used in combination with alpha-emitting radionuclides (Zechmann et al. Eur J Nucl Med Mol Imaging. 2014;41 (7):1280-92 and Kratochwil et al. J Nucl Med. 201 7 Apr 1 3. pii : jnumed.1 1 7.1 91 395. doi: 1 0.2967/jnumed.1 1 7.191 395 [Epub]). One possible solution to that problem involves the use of PSMA-binding agents with a high-affinity towards PSMA (Kratochwil et al. J Nucl Med. 201 5; 293-298 and Chatalic et al. Theragnostics. 201 6; 6: 849- 861 ).
Recently, Kel ly et al. 0 Nucl Med. 201 7 pii : jnumed.1 1 6.1 88722. doi: 1 0.2967/jnumed.1 1 6.1 88722. [Epub ahead of print]) evaluated agents exhibiting affinity for both PSMA and for human serum albumin (HSA). The ligands developed by Kelly et al. comprise a >(iodophenyl)butyric acid entity for HSA binding and an urea-based PSMA binding entity. In the compounds developed by Kel ly et al., radiotherapeutic iodine (131 l) is covalently attached to the HSA binding moiety, which is in turn directly connected to the PSMA binding entity via a hydrocarbyl chain. However, the evaluated compounds are considerably limited in terms of the applied radionuclide which is limited to iodine. Further, no improved internal ization/uptake in target cells was demonstrated for the evaluated compounds. Another approach was followed by Choy et al. Theranostics 201 7; 7(7):1 928-1 939, who evaluated 177Lu-labeled phosphoramidate-based PSMA inhibitor with an albumin- binding entity. A DOTA chelator complexing the 177Lu radionuclide was ether-linked to the irreversible PSMA inhibitor CTT1 298 (EP 2970345 A1 ). Phosphoramidate-based PSMA binding motive, however, exhibits only poor stabi lity, especial ly at elevated temperatures (elevated temperatures under extended acidic conditions lead to hydrolysis of phosphoramidate P-N bond), which are required for the coordinative radiolabeling reaction via chelators such as DOTA. Therefore a direct radiolabeling reaction cannot be applied and a multi-step pre-labeling approach has to be used. Thus, 177Lu-DOTA-azide as precursor should be prepared; subsequently the precursor has to be coupled to a dibenzocyclooctyne- derivatized PSMA motive. Finally, elaborate HPLC purification of the coupled compound must be undertaken; reformulation with evaporation (under N2 atmosphere) of the HPLC- eluent and dissolving in a physiological medium need to be performed. This procedure is likely not possible for a clinical application when high activities are being produced. Pre- clinical biodistribution data demonstrate poor performance of the radiolabeled agent especially regading tumour-to-kidney ratios which did not exceed far above 1 .
Despite advances over the years, diagnosis and management of prostate cancer still remains challenging. New diagnostic or imaging agents capable of targeting PCa tumor cells in a highly selective manner and exhibiting favorable pharmacoki netic properties for rapid and non-invasive tumor visualization and therapy are needed to enable early detection and treatment of PCa. It is thus an object of the present invention to overcome the disadvantages in the prior art and comply with the need in the art.
That object is solved by the subject-matter disclosed herein, more specifically as set out by the claim set.
General comments
Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. In the following, the elements of the present invention wi ll be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined i n any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the expl icitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Throughout this specification and the claims which follow, unless the context requires otherwise, the term„comprise", and variations such as„comprises" and„comprising", wi ll be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step. The term „consist of" is a particular embodiment of the term„comprise", wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term„comprise" encompasses the term „consist of". The term „comprising" thus encompasses „including" as well as „consisting" e.g., a composition„comprising" X may consist exclusively of X or may include something additional e.g., X + Y.
The terms „a" and „an" and „the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herei n or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
The word substantially" does not exclude„completely" e.g., a composition which is substantially free" from Y may be completely free from Y. Where necessary, the word substantially" may be omitted from the definition of the invention.
The term„about" in relation to a numerical value x means x + 1 0%.
In the present invention, if not otherwise indicated, different features of alternatives and embodiments may be combined with each other.
For the sake of clarity and readability the followi ng definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.
Definitions The term „hydrocarbyl" refers to residues of hydrocarbon groups, i.e., hydrocarbon chain radicals, preferably independently selected from the group alkyl, alkenyl, alkynyl, aryl and aralkyl.
The term „alkyl" comprises linear („straight-chain"), branched and cyclic chain radicals having 1-30 carbon atoms, preferably 1-20, 1-15, 1-10, 1-8, 1-6, 1-4, 1-3 or 1-2 carbon atoms. For instance, the term „Ci-i2 alkyl" refers to a hydrocarbon radical whose carbon chain is straight-chain or branched or cyclic and comprises 1 to 12 carbon atoms. Specific examples for alkyl residues are methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl or triacosyl, including the various branched-chain and/or cyclic isomers thereof, e.g. tert.-butyl or isopentyl, and so on. Cyclic alkyl isomers are also referred to as „cycloalkyl" herein to refer to saturated alicyclic hydrocarbons comprising 3 ring carbon atoms. Substituted" linear, branched and cyclic alkyl groups are generally also encompassed by the term. The term further includes„heteroalkyl", referring to alkyl groups wherein one or more C-atoms of the carbon chain are replaced with a heteroatom such as, but not limited to, N, O, and S. Accordingly, the term further includes „heterocyclyl" or „heterocycloalkyl", referring to non-aromatic ring compounds containing 3 or more ring members, of which one or more ring carbon atoms are replaced with a heteroatom such as, but not limited to, N, O, and S. Heterocyclyl groups encompass unsaturated, partially saturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups. Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl,azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[ 1 ,3 ]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazol inyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Heterocyclyl groups may be substituted or unsubstituted. Representative substituted heterocyclyl groups may be monosubstituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above.
The term„cyclic" includes the term„polycyclic", referring to structures having more than one ring structure. In particular, the term„cyclic" also refers to spirocyclic structures, wherein two or more rings have one atom in common, and 5 fused polycyclic structures, wherein two or more rings have at least two atoms in common.
The term„alkenyl" as employed herein comprises linear, branched and cyclic chain 1 0 radicals having 2-30 carbon atoms, preferably 2-20, 2-1 5, 2-1 0, 2-8, 2-6, 2-4, or 2-3 carbon atoms, including at least one carbon-to-carbon double bond. Specific examples of „alkenyl" groups are the various alkenic unsaturated equivalents of those given with respect to alkyl groups, named after the conventions known to the person ski lled in the art, depending on the number and location of carbon-to-carbon double bond or bonds, e.g. butanediylidene, 1 -propanyl-3-ylidene.„Alkenyl" groups preferably contain at least 1 , more preferably at least 2, 3, 4, 6, 7, 8, 9, 1 0, 1 1 , 1 2, 1 3, 14, 1 5, or 1 6 double bonds, wherein a double bond is preferably located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 12, 1 3, 1 4, 1 5, 1 6, 1 7, 1 8, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 of the hydrocarbyl chain. Alkenyl groups may be substituted or unsubstituted. The term„alkynyl" as employed herein comprises straight, branched and cyclic chain radicals having 2-30 carbon atoms, preferably 2-20, 2-1 5, 2-1 0, 2-8, 2-6, 2-4, or 2-3 carbon atoms, including at least one carbon-to-carbon triple bond. Specific examples of „alkynyl" groups are the various alkynic unsaturated equivalents of those given with respect to alkyl and alkenyl groups, named after the conventions known to the person ski lled in the art, depending on the number and location of carbon-to-carbon triple bond or bonds.„Alkynyl" groups preferably contain at least 1 , more preferably at least 2, 3, 4, 6, 7, 8, 9, 1 0, 1 1 , 1 2, 1 3, 14, 1 5, or 1 6 triple bonds, wherein a double triple bond is preferably located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 12, 1 3, 14, 1 5, 1 6, 1 7, 30 1 8, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 of the hydrocarbyl chain. Alkynyl groups may be substituted or unsubstituted.
The term„aryl" refers to monocyclic or polycyclic or fused polycyclic aromatic ring systems. The term includes monocyclic or polycyclic or fused polycyclic aromatic „heteroaryl" ring systems wherein at least one carbon atom of the ring system is substituted by a heteroatom. Typically, the terms„aryl" and„heteroaryl" refers to groups having 3-30 carbon atoms., such as 3-10, in particular 2-6 carbon atoms.
The terms„arylalkyl" or„aralkyl" are used interchangeably herein to refer to groups comprising at least one alkyl group and at least one aryl group as defined herein. In an aralkyi group as defined herein, the aralkyi group is bonded to another moiety of the compounds or conjugates of the invention via the alkyl group as exemplified by a benzyl group.
The term„halogen" or„halo" as used herein includes fluoro (F), chloro (CI), bromo (Br), iodo (I).
The term„heteroatom" includes N, O, S and P, preferably N and O.
The term substituted" refers to a hydrocarbyl group, as defined herein (e.g., an alkyl or alkenyl group) i n which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted" group will be substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1 , 2, 3, 4, 5, or 6 substituents. Examples of substituent groups i nclude: halogens (i.e., F, CI, Br, and I); hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls ( oxo ); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i.e., CN), haloalkyl, aminoalkyl, hydroxyalkyl, cycloalkyl and the like.
Conjugates
The present invention provides novel plasma protein-binding PSMA ligands with improved tumor targeting properties and favorable pharmacokinetic profi les. As used herein, the term pharmacoki netics" preferably includes the stabi lity, bioavailability, absorption, biodistribution, biological half-life and/or clearance of a therapeutic or diagnostic agent in a subject. The present inventors provided novel conjugates by covalently coupling a PSMA- peptidomimetic urea-based binding entity via suitable spacers and linkers to a a chelator capable of complexing therapeutic/diagnostic radionuclides on the one hand, and a human serum albumin (HSA) binding entity on the other hand. The spacer and linker groups connecting the binding entities and chelator were found to be crucial for the targeting and pharmacokinetic properties of the resulting conjugates. The novel conjugates preferably exhibit superior and specific cellular uptake and internalization characteristics. The inventors demonstrated that the HSA binding entity advantageously effected (1 ) compartmentalization of the conjugates in the blood (where off-target effects in healthy tissues are limited, without compromising access to the tumor vasculature), (2) extended blood clearance, and (3) increased tumor uptake and retention (by increasing the number of passes through the tumor bed). Introduction of a HSA binding entity thereby advantageously improves biodistribution and, eventually, therapeutic efficacy of the inventive compounds. In particular, the conjugates provided herein advantageously exhibit an increased tumor uptake as compared to other PSMA ligands known in the art. The conjugates' favourable tumor uptake characteristics in particular al low reducing the administered activity to achieve the desired dose for a therapeutic effect or sufficient uptake allowing imaging (diagnosis). To that end, the conjugates are commonly provided in the form of radiolabeled complexes with the chelator complexing a therapeutic and/or diagnostic radionuclide (often a metal isotope). A decrease in the required dose of the novel conjugates (and in particular their radiolabeled (metal) complexes) inter alia has the fol lowing advantages: (1 ) a lower quantity of radionuclides (radioactivity) is required (resulting in lower manufacturing costs, better availabi lity -both are particularly relevant i n case of alpha-emitters such as e.g. 225Ac which are difficult to produce and costly - and preferably a longer shelf-life due to a decreased self-irradiation which commonly results in degradation of radiolabeled complexes (i.e. radiolysis); (2) the patient is subjected to a lower total absorbed dose of irradiation (preferably rendering ambulant treatment possible, and placing a lower burden on the environment).
The inventive conjugates are thus promising theragnostic agents with optimal characteristics both for nuclear imaging and endoradiotherapy.
Generally, the novel PSMA l igands according to the invention (also referred to as „conjugates" or "compounds" herein) thus include a first terminal group (a chelating agent), a second terminal group (an albumin binding entity) and a third terminal group (a PSMA binding entity) that are covalently connected or linked to each other via appropriate linkers or spacers.
In a first aspect, the present invention relates to a compound of General Formula (1 ):
Linker Pbm
Figure imgf000013_0001
Spacer Abm
(1 ) wherein
chelator, preferably as defined herein
Abm is an albumin binding entity, preferably as defi ned herein,
Pbm is a PSMA binding entity, preferably as defined herein,
the spacer comprises at least one C-N bond,
the linker is characterized by General Formula (6) as defined herein,
integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0, and
the -CH- group i n General Formula (1 ) is a "branching point" connecting the PSMA binding entity (Pbm) and the albumin binding entity (Abm), or pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof. D, Abm, Pbm, Linker and Spacer are preferably defined as described herein.
Specifical ly, the present invention provides compounds according to General Forrr (D(i) or (D(ii):
R3
Linker N N H
H
N— [CH2]a CH X
Spacer N H Abm
R5
(D(i)
Linker Pbm
\
cer
Figure imgf000014_0001
(D(ii) wherein
Abm is an albumin bi nding entity, preferably as defined herein, Pbm is a PSMA bi nding entity, preferably as defi ned herein, D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2 -(4,7, 1 0-tris(carboxymethyl)-1 ,4,7,1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-1 -[methyl(2-carboxyethyl)- phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 1 5-tetraazabicyclo[9,3, 1 ]pentadeca-1 (1 5),1 1 , 1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylen- etriaminepentaacetic acid (DTPA), or derivatives thereof,
X is each independently selected from O, N, S or P,
R1 and R2 are each independently selected from H, F, CI, Br, I, branched, unbranched or cyclic C1-C12 hydrocarbyl, C2-C12 alkenyl, C2-C12 alkylnyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, CONR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, or R1 and R2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic G-Go hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optionally substituted by up to 3 groups independently selected from F, CI, Br, I, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, and SR7, =0, =S and =NH,
Y is selected from a single bond or a linear, branched or cyclic, optional ly substituted G-G2 alkyl, optionally interrupted by up to two heteroatoms, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, wherein one or more of the non-adjacent CH2-groups may independently be replaced by -0-, -CO- -CO-O-, -O-CO- -NR6-, -NR6-CO- -CO-NR6-, -NR6-COO- - O-CO-NR6-, -NR6-CO-NR6-, -CH=CH- , -C≡C- -O-CO-O-, SR6-, S03R6-,
R6 and R7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl,
R3, R4 and R5 are each independently selected from -COH, -C02H, -SO2H, - SO3H, -SO4H, -PO2H, -PO3H, -PO4H2, -C(0)-(G-Go)alkyl, -C(O)-O(G-G0)alkyl, - C(0)-NHR8, or -C(0)-NR8R9' wherein R8 and R9 are each independently selected from H, bond, (G -Go)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(Ci-Cio)alkylene, -(CH2)P-NH, -(CH2)p-(Ci-Ci0)alkyene, -(CH2)P-NH-C(0)-(CH2)q/ -(CHrCH2),-NH-C(0)-(CH2)p, -(CH2)p-CO-COH, -(CH2)p-CO-C02H, -(CH2)P-C(0)NH- C[(CH2)q-COH]3, -C[(CH2)p-COH]3, -(CH2)p-C(0)NH-C[(CH2)q-C02H]3 -C[(CH2)P- C02H]3 or -(CH2)p-(C5-Ci4)heteroaryl,
the spacer comprises at least one C-N bond,
the linker is characterized by General Formula (6) as defined herein, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0,
or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes
It is particularly envisaged that the structure highlighted in Formula (1 ) below comprises at least one peptide bond:
Figure imgf000016_0001
The inventive conjugates are ligands exhibiting affinity towards both PSMA and HSA. The term "ligand" as used herein refers to a compound capable of interacting with (targeting, binding to) a target (here: PSMA or HSA). The inventive conjugates may also be defined functional ly as "PSMA targeting agents". Preferably, "ligands" are capable of selectively binding to their target. The term "selectively binding" means that a compound binds with a greater affinity to its intended target than it binds to another, non-target entity.
"Binding affinity" is the strength of the bi nding interaction between a ligand (e.g. a smal l organic molecule, protein or nucleic acid) to its target/binding partner. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), a ratio of the "off-rate" (k0ff) and the "on-rate" (kon), which is used to evaluate and rank order strengths of bi molecular interactions. The "on-rate" (Kon) characterizes how quickly a ligand binds to its target, the "off-rate" (Koff) characterizes how quickly a ligand dissociates from its target. KD (Koff Kon) and binding affinity are inversely related. Thus, the term "selectively binding" preferably means that a ligand binds to its intended target with a KD that is lower than the KD of its binding to another, non-target entity. There are many ways to measure binding affinity and dissociation constants, such as ELISA, gel-shift assays, pull-down assays, equi librium dialysis, analytical ultracentrifugation, surface plasmon resonance, and spectroscopic assays.
In the context of the present invention, the KD for binding of the PSMA binding entity (HSA binding entity) to a non-target entity may be at least 1 .5-fold, preferably at least 2-, 3-, 5-, 10-, 1 5-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 1 00- 200-, 300-, 400-, 500-, 750-, or 1000-fold the KD for binding of said conjugate or moiety to human PSMA (HSA).
In the context of the present invention, it may further be preferred that the conjugates bind to PSMA with high binding affinity with KD values in the nanomolar (nM) range and with moderate affinity to HSA in the micromolar range (μΜ (micromolar)). Specifically, it may be preferred to balance the PSMA and HSA-binding affinities so as to increase tumor uptake and retention and extend blood clearance, while reducing potentially damaging off-target effects. In particular, the inventive conjugates may exhibit a higher binding affinity towards PSMA than towards HSA.
A l b u m i n b i n d i n g e n ti ty The inventive conjugates comprise an (additional -as compared to known PSMA ligands) albumin binding entity (also referred to as an "albumin binding moiety") as described herein, which is preferably capable of selectively binding to human serum albumin (HSA). The term "selectively binding" is defined above.
In particular, the present invention provides compounds according to General Formula (1 )(i):
Figure imgf000018_0001
(D(i) wherein
Abm is an albumin binding entity,
D is a chelator, preferably selected from 1 ,4,7,1 O-tetraazacyclododecane- 1 ,4,7,1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOT A), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2 -(4,7, 1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-1 -[methyl(2-carboxyethyl)- phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 1 5-tetraazabicyclo[9,3, 1 .]pentadeca-1 (1 5), 1 1 , 1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and
Diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,
X is selected from O, N, S or P,
R3, R4 and R5 are each independently selected from -COH, -CC½H, -SO2H, - SO3H, -SO4H, -PO2H, -PO3H, -P04H2, -C(O)-(Ci-Ci0)alkyl/ -C(O)-O(Ci-Ci0)alkyl, - C(0)-NHR8, or -C(0)-NR8R9, wherein R8 and R9 are each independently selected from H, bond, (Ci-Cio)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(Ci-Cio)alkylene, -(CH2)P-NH, -(CH2)p-(Ci-C10)alkyene, -(CH2)p-NH-C(0)-(CH2)q, -(CHrCH2)rN H-C(0)-(CH2)p, -(CH2)p-CO-COH/ -(CH2)p-CO-C02H, -(CH2)P-C(0)NH- C[(CH2)q-COH]3, -C[(CH2)p-COH]3, -(CH2)p-C(0)NH-C[(CH2)q-C02H]3, -C[(CH2)P- C02H]3 or -(CH2)p-(C5-Ci 4)heteroaryl/
the spacer comprises at least one C-N bond, and
the linker is characterized by General Formula (6) as defined herein, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0, or a pharmaceutical ly acceptable salt, ester, solvate or radiolabeled complex thereof.
The albumin binding entity (Abm) may be any albumin binding entity. Particularly preferred albumin binding entities are described herein below. The albumin binding entity may preferably bind non-covalently to serum albumin, preferably HSA, typically with a binding affinity of less than about 100 μΜ (micromolar), e.g. of about 3 μΜ (micromolar) to 50 μΜ (micromolar).
Human Serum Albumin (HSA) is the most abundant protein in human plasma and constitutes about half of serum protein. The term "Human Serum Albumin" or "HSA" as used herein preferably refers to the serum albumin protein encoded by the human ALB gene. More preferably, the term refers to the protein as characterized under UniProt Acc. No. P02768 (entry version 240, last modified May 1 0, 201 7, or functional variants, isoforms, fragments or (post-translationally or otherwise modified) derivatives thereof. Without wishing to be bound by specific theory, it is hypothesized that the albumin binding entity (Abm) of the inventive conjugates preferably extends circulation half-life of the conjugates, and effects compartmentalization of the inventive conjugates in the blood and improved delivery to the PSMA-expressing (tumor) target cel ls or tissues, resulting in increased tumor: non-target ratios for PSMA expressing normal (non-tumorous) organs (like kidneys, lacrimal glands, and salivary glands). The albumin binding entity is thus envisaged to confer improved pharmacokinetic properties to the inventive conjugate, preferably without interfering with (reduci ng or abolishing) the desired function of the chelating agent and the PSMA binding entity. In terms of structure, typical albumin binding entities in accordance with the present invention may preferably comprise linear and branched lipophilic groups comprising 1-40 carbon atoms and a distal acidic group. Suitable albumin binding entities are inter alia described in US 2010/172844 A1, WO 2013/024035 A1 and WO 2008/053360 A2, which are incorporated by reference in their entirety herein.
In accordance with the above, in the conjugates of the present invention, the albumin binding entity is preferably characterized by General Formula (2):
Figure imgf000020_0001
(2) wherein
R1 and R2 are each independently selected from H, F, CI, Br, I, branched, unbranched or cyclic Ci-G2 hydrocarbyl, C2-C12 alkenyl, C2-O2 alkylnyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, CONR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, or R1 and R2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic G-Go hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optionally substituted by up to 3 groups independently selected from F, CI, Br, I, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, and SR7, =0, =S and =NH.
Y is selected from a single bond or a linear, branched or cyclic, optionally substituted C1-G2 alkyl, optionally interrupted by up to two heteroatoms, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, wherein one or more of the non-adjacent CH2-groups may independently be replaced by -0-, -CO- -CO-O-, -O-CO-, -NR6-, -NR6-CO- -CO-NR6-, -NR6-COO- - O-CO-NR6-, -NR6-CO-NR6-, -CH=CH- , -C≡C- -O-CO-O-, SR6-, SO3R6-,
R6 and R7 are each independently selected from H or branched, unbranched or cyclic G.12 hydrocarbyl, and X is selected from O, N, P or S. R1 and R2 may be in ortho-, meta or para-position.
When R1 and R2 are joined in order to, together, form a cyclic structure, said cyclic structure is preferably a linear or branched hydrocarbyl chain of 3-1 2, more preferably 3-1 0, even more preferably 3-9, 3-8, 3-7, 3-6, 3-5, 3-4 or 4 carbon atoms bonded at two positions to the phenyl ring, i.e. forming two bonds to said phenyl ring, such as to form a ring structure fused to said phenyl ring. Specifically, said cyclic structure may be selected from (substituted or unsubstituted) adamantyl. Preferably, said two bonds are preferably situated at the meta (3-) and para (4-) positions, at the ortho (2-) and meta positions or at the ortho and para positions of said phenyl ring. Said cyclic structure is optionally interrupted by up to 2, preferably 1 or none heteroatoms. Preferably, said cyclic structure may be a C4 chain fragment (1 ,4-diradical) linked by its 1 - and 4- atoms to said phenyl ring to form a six-membered ring fused to said phenyl ring, preferably at the meta and para positions of said phenyl ring, i.e., preferably forming a meta- and para-fused six-membered ring.
Preferably, R1 and R2 may each be independently selected from H, halogen, preferably iodine or bromine, and G-6 alkyl, preferably C1-3 alkyl, even more preferably methyl. More preferably, R1 is H and R2 is selected from halogen, preferably iodine or bromine, and G-6 alkyl, preferably G-3 alkyl, even more preferably methyl. Even more preferably, R1 is H and R2 is H or is in the para position and selected from iodine, bromine and methyl.
Preferably, Y may be a linear or branched, optionally substituted, G-C12 hydrocarbyl, more preferably a linear or branched, optionally substituted, G-Go hydrocarbyl, even more preferably a linear or branched, optionally substituted, G-G hydrocarbyl, even more preferably a a linear or branched, optionally substituted, G-G hydrocarbyl.
Most preferably, Y may be -(CH2)3-.
Preferably, X may be O.
Accordingly, the albumin binding entity according to Formula (2) may preferably comprise or consist of any one of Formulae (2a)-(2c):
Figure imgf000022_0001
Figure imgf000022_0002
(2 c)
Other possible -potentially less preferred- albumin binding entities are disclosed inter alia in US 2010/01 72844 A1 .
S pa ce r In the i nventive conjugates, the albumin binding entity is conjugated (i.e. covalently linked or attached to) to the -CH- "branching point" via a "spacer". The term "spacer" is used herein to specifically refer to the group connecting and spanning the distance between the albumin binding entity and the -CH- "branching point", and/or "spacing" these groups apart from the remaining groups/entities of the conjugate.
The spacer may preferably avoid sterical hindrance between the albumin bindi ng entity and the other groups or entities of the inventive conjugate and ensure sufficient mobility and flexibi lity. Futher, the spacer may preferably be designed so as to confer, support and/or allow sufficient HSA binding, high affinity PSMA binding, and rapid and optional ly selective penetration of PSMA positive cells through internalization of the PSMA-conjugate complex.
The present inventors determined that the spacer should preferably comprise at least one C-N bond. Suitable spacers should preferably be stable in vivo. Spacer design may typically depend on the overall conjugate and may preferably be chosen to promote the functionality of the remaining conjugate (e.g. PSMA binding, HSA binding, internalization etc.). Accordingly, spacers may be for instance be rigid or flexible, influencing either lipophilicity or hydrophilicity of the overall conjugate, and so on.
Preferably, the spacer may comprise a li near or branched, optionally substituted G- C20 hydrocarbyl comprising up to 5 heteroatoms, more preferably G -C12 hydrocarbyl, even more preferably C2-C6 hydrocarbyl, even more C2-C4 hydrocarbyl. The hydrocarbyl may preferably comprise at least one, optionally up to 4 heteroatoms preferably selected from N.
Preferably, the spacer may be -[CH 10]U-N 11-, wherein R10 and R11 may each be independently selected from H and branched, unbranched or cyclic C1-G2 hydrocarbyl and wherein u may be an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0. More preferably, R10 and R11 may be H, and u may be an integer selected from 2, 3 or 4. Most preferably, R10 and R1 ' may be H and u may be 4.
Accordingly, the inventive conjugates may preferably comprise a spacer of Formula
(3a):
Figure imgf000023_0001
(3a)
Accordingly, preferred conjugates according to the invention (e.g. PSMA-ALB-03 and PSMA-ALB-06 evaluated in the appended examples), comprise an albumin bi nding entity of Formula (2a)-(2c) connected to the "branching point" via a spacer of Formula (3a).
Alternatively or additionally, the spacer may comprise at least one amino acid residue. As used herein, the term "amino acid residue" refers to a specific amino acid monomer as a moiety within the spacer. An "amino acid" is any organic molecule comprising both an acidic (typically carboxy (-COOH)) and an amine (-NH2) functional group. One or both of said groups may optionally be derivatized. The amino and the acidic group may be in any position relative to each other, but amino acids typically comprise 2-amino carboxylic acids, 3-amino carboxylic acids, 4- amino carboxylic acids, etc. The amine group may be attached to the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th (etc.) up to t he 20th carbon atom of the amino acid(s). In other words, the amino acid(s) may be (an) alpha-, beta-, gamma-, delta-, epsilon- (etc.) up to an omega-amino acid(s). Preferably, the acidic group is a carboxy (-COOH) group. However, other acidic groups selected from -OPO3H, -PO3H, -OSO3H or-SOsH are also conceivable.
Preferably, the amino acid residue(s) is/are derived from naturally occurring amino acid(s), or derivatives thereof. It is further preferred that the amino acid residues(s) is/are derived from alpha (oc-)amino acid(s), wherein the amino acid(s) may be (a) D- or L-amino acid(s).
More preferably, said amino acid(s) is/are the D- or the L- enantiomer of an amino acid selected from the group arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and/or valine.
Most preferably, said amino acid(s) is/are (D-/L-) aspartate, glutamate or lysine. The spacer may comprise 1, 2, 3, 4 or 5 amino acid residue(s), in particular D-aspartate, D- glutamate or L-Lysine residues. In conjugates comprising the D-enantiomer, the use of the D- enantiomer may provide the beneficial effect of further reducing the rate of metabolisation and thus clearance from the bloodstream. Preferably, the spacer may comprise between 2 and 3 of such amino acid residues in particular D-aspartate or D-glutamate residues. In other words, the spacer may comprise a peptide, which preferably consists of 2 to 5 amino acids, more preferably of 2 to 3 amino acids. Alternatively, the spacer may comprise between 1 and 2 amino acids selected from L-Lysine.
Accordingly, the inventive conjugates may comprise a spacer of Formula (3b):
Figure imgf000025_0001
wherein
m is an integer selected from 1 or 2,
n is an integer selected from 1 , 2, 3, 4 or 5, preferably from 2 or 3.
Alternatively, the spacer may comprise an amino acid residue connected to th "branching point" via a linear or branched, optional ly substituted, G-C20 hydrocarbyl grou comprising at least one N heteroatom.
Accordingly, the inventive conjugates may comprise a spacer of Formula (3c):
Figure imgf000025_0002
(3c) wherein o is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0. Preferably, o may be 5.
C he l ato r
The inventive conjugates further comprise a chelator.
The terms„chelator" or„chelating moiety" are used interchangeably herein to refer to polydentate (multiple bonded) ligands capable of forming two or more separate coordi nate bonds with (coordinating") a central (metal) ion. Specifically, such molecules or molecules sharing one electron pair may also be referred to as„Lewis bases". The central (metal) ion is usually coordinated by two or more electron pairs to the chelating agent. The terms, „bidentate chelating agent",„tridentate chelating agent", and„tetradentate chelating agent" are art-recognized and refer to chelating agents having, respectively, two, three, and four electron pairs readily available for simultaneous donation to a metal ion coordinated by the chelating agent. Usually, the electron pairs of a chelating agent forms coordinate bonds with a single central (metal) ion; however, in certain examples, a chelating agent may form coordinate bonds with more than one metal ion, with a variety of binding modes being possible.
The terms coordinating" and coordination" refer to an interaction in which one multi-electron pair donor coordinatively bonds (is coordinated") to, i.e. shares two or more unshared pairs of electrons with, one central (metal) ion.
The chelating agent is preferably chosen based on its ability to coordinate the desired central (metal) ion, usually a radionuclide as specified herein.
Accordingly, the chelator D may be characterized by one of the fol lowing Formulas (4a)-(4jj):
Figure imgf000026_0001
HBED (4d) HBED-CC TFP (4e) h DEPDPA (4f)
Figure imgf000027_0001
DFO-B (4g) Deferiprone (4h) CP256 (4i)
Figure imgf000027_0002
YM103 (4j) R =H TETA CB-TE2A (4m)
R =CH2C02H (4k)
TE2A (4I)
Figure imgf000027_0003
R =H Sar (4n) R =H TRAPH (4p) NOPO (4t) R =NH2 DiAmSar (4o) R = (CH2)2C02H TRAP-Pr
R =CH2OH (4q)
R =phenyl TRAP-OH
(4r)
TRAP-Ph
(4s)
Figure imgf000028_0001
DEDPA (BCPE) (4u) PCTA (4v)
Figure imgf000028_0002
DTPA (4dd) EDTMP (4ee) AAZTA (4ff)
Figure imgf000029_0001
DOTAGA
DOTAGA 4gg) D03AP(4hh) D03APPrA (4ii)
Figure imgf000029_0002
D03APABn (4jj)
Preferably, the chelator may be DOTA (1 ,4,7, 1 0-tetraazacyclododecane-1 ,4,7, 1 0- tetraacetic acid, which may be characterized by Formula (4a)), NODAGA (2 -(4,7- bis(carboxymethyl)-1 ,4,7-triazonan-1 -yl)-pentanedioic acid, which may be characterized by Formula (4c)), or derivatives thereof. Advantageously, DOTA effectively forms complexes with diagnostic (e.g. 68Ga) and therapeutic (e.g. 90Y or l 77Lu) radionuclides and thus enables the use of the same conjugate for both imaging and therapeutic purposes, i .e. as a theragnostic agent. DOTA derivatives capable of complexing Scandium radionuclides (43Sc, 44Sc, 47Sc), including D03AP (which may be characterized by Formula (4hh)), D03APPrA (which may be characterized by Formula (4ii)), or D03APABn (which may be characterized by Formula (4jj)) may also be preferred and are described in Kerdjoudj et al. Dalton Trans., 201 6, 45, 1 398- 1409.
Other preferred chelators in the context of the present invention include N ,N" -bis[2- hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7- triazacyclo-nonane-1 ,4,7-triacetic acid (NOTA), 2-(4,7, 1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetra-azacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydo-nonane-1 -[methyl(2-carboxyethyl)-phosphinic acid]-4,7-bis-[methyl(2-hydroxymethyl)-phosphi nic acid] (NOPO),3,6,9, 1 5-tetra- azabicyclo[9,3, 1 ]-pentadeca-1 (1 5), 1 1 , 1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5- [Acetyl(hydroxy)amino]-pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}- amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylene-triaminepentaacetic acid (DTPA).
The chelator group, for example, the DOTA group may be complexed with a central (metal) ion, in particular a radionuclide as defined herein. Alternatively, the chelator group, for example DOTA, may not be complexed with a central (metal) ion, in particular a radionuclide as defined herein, and may thus be present in uncomplexed form. In cases where the chelator (e.g. DOTA) is not complexed with said metal ion, the carboxylic acid groups of the chelator can be in the form of a free acid, or in the form of a salt. PSMA b i n d i n g e nti ty
The inventive conjugates comprise a PSMA binding entity (also referred to as "PSMA binding moiety") herein, which is preferably capable of selectively binding to human PSMA. The term "selectively binding" is defined above. In particular, the present invention provides compounds according to General
Formula (1 )(ii):
Figure imgf000030_0001
(1 )(ϋ) wherein
Pbm is a PSMA binding entity,
D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2-(4,7, 1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-1 -[methyl(2-carboxyethyl)- phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 1 5-tetraazabicyclo[9,3,1 .]pentadeca-1 (1 5),1 1 , 1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylene- triaminepentaacetic acid (DTPA), or derivatives thereof,
X is O, N, S or P,
R1 and R2 are each independently selected from H, F, CI, Br, I, branched, unbranched or cyclic C1-G2 hydrocarbyl, C2-C12 alkenyl, C2-G2 alkylnyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, CONR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, or R1 and R2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic G-Go hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optional ly substituted by up to 3 groups independently selected from F, CI, Br, I, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, and SR7 , =0, =S and =NH,
Y is selected from a single bond or a linear, branched or cyclic, optionally substituted G-G2 alkyl, optionally interrupted by up to two heteroatoms, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, wherein one or more of the non-adjacent CH2-groups may independently be replaced by -0-, -CO- -CO-O-, -O-CO-, -NR6-, -NR6-CO- -CO-NR6-, -NR6-COO- - O-CO-NR6-, -NR6-CO-NR6-, -CH=CH- , -OC-, -O-CO-O-, SR6-, SO3R6-,
R6 and R7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl, the spacer comprises at least one C-N bond,
the linker is characterized by General Formula (6) as defined herein, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0, or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof.
The PSMA binding entity may bind reversibly or irreversibly to PSMA, typical ly with a binding affinity less than about 1 00 μΜ (micromolar).
Human Prostate-specific membrane antigen (PSMA) (also referred to as glutamate carboxypeptidase II (GCPII), folate hydrolase 1 , folypoly-gamma-glutamate carboxypeptidase (FGCP), and N-acetylated-alpha-linked acidic dipeptidase I (NAALADase I)) is a type II transmembrane zinc metal lopeptidase that is most highly expressed in the nervous system, prostate, kidney, and small intestine. It is considered a tumor marker in prostate cancer. The term "Human Prostate-specific membrane antigen" or "PSMA" as used herein preferably refers to the protein encoded by the human FOLH1 gene. More preferably, the term refers to the protein as characterized under UniProt Acc. No. Q04609 (entry version 1 86, last modified May 1 0, 201 7, or functional variants, isoforms, fragments or (post-translational ly or othweise modified) derivatives thereof.
The PSMA-binding entity may generally be a binding entity capable of selectively (and optionally irreversibly) binding to (human) Prostate-Specific Membrane Antigen (cf. Chang Rev Urol. 2004; 6(Suppl 1 0): S1 3-S1 8).
The PSMA binding entity is preferably chosen by its abi lity to confer selective affinity towards PSMA. Preferred PSMA binding moieties are described in WO 201 3/022797 A1 , WO 201 5/05531 8 A1 and EP 2862857 A1 , which are incorporated by reference in their entirety herein.
Accordingly, in the conjugates of the present invention, the PSMA binding entity may preferably be characterized by General Formula (5):
Figure imgf000033_0001
(5) wherein
X is selected from O, N, S or P,
R3, R4 and R5 are each independently selected from -COH, -C02H, -SO2H, -SO3H, - SO4H, -P02H, -PO3H, -P04H2/ -C(O)-(Ci-Ci0)alkyl, -C(O)-O(Ci-C10)alkyI, -C(0)-NHR8, or - C(0)-NR8R9' wherein R8 and R9 are each independently selected from H, bond, (C1- C10)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(G- Cio)alkylene, -(CH2)P-NH, -(CH2)P-(C,-Cio)alkyene, -(CH2)p-NH-C(0)-(CH2)q -(CHrCH2)t-NH- C(0)-(CH2)p, -(CH2)p-CO-COH, -(CH2)p-CO-C02H, -(CH2)p-C(0)NH-C[(CH2)q-COH]3, - C[(CH2)p-COH]3, -(CH2)p-C(0)NH-C[(CH2)q-C02H]3, -C[(CH2)p-C02H]3 or -(CH2)P-(C5- Ci4)heteroaryl, and
b, p, q, r, t is each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In preferred PSMA binding entities, b may be an integer selected from 1, 2, 3, 4 or 5, R3, R4 and R5 may each be C02H, X may be O. Li n ker
In the inventive conjugates, the PSMA binding entity is attached/connected to the - CH- "branching point" via a suitable linker. The term "linker" is used herein to specifically refer to the group connecting or linking and thus spanning the distance between the PSMA binding entity and the -CH- "branching point", and/or„spacing" the PSMA binding entity apart from the remaining conjugate.
The linker may preferably avoid sterical hindrance between the PSMA binding entity and the other groups or entities of the inventive conjugate and ensure sufficient mobi lity and flexibility. Futher, the linker may preferably be designed so as to confer, support and/or allow sufficient HSA binding, high affinity PSMA binding, and rapid and optionally selective penetration of PSMA positive cells through internalization of the PSMA-conjugate complex.
PSMA binding entities, and in particular preferred PSMA binding entities of General Formula (5), may preferably be linked to the inventive conjugate via a suitable linker as described, e.g. in EP 2 862 857 A1 . Said linker may preferably confer optimized lipophi lic properties to the inventive conjugate to increase PSMA binding and cellular uptake and internalization. The linker may preferably comprise at least one cyclic group and at least one aromatic group (in particular in group Q and W).
Accordingly, in the inventive conjugates, preferred linkers may be characterized by General Formula (6):
Figure imgf000034_0001
(6) wherein
X is each i ndependently selected from O, N, S or P,
Q is selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl, preferably from substituted or unsubstituted C5-G4 aryl, C5-Ci4 alkylaryl or C5-C14 cycloalkyl, W is selected from -(CH2)c-aryl or -(CH2)c-heteroaryl, wherein c is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 . Without wishing to be bound by specific theory, it is thought that hydrophilic or polar functional groups within or pendant from the linker (in particular Q, W) may advantageously enhance the PSMA-binding properties of the inventive conjugate. Where Q is a substituted aryl, alkylaryl or cycloalkyl, exemplary substituents are listed in the "Definitions" section above and include, without limitation, halogens (i.e., F, CI, Br, and I); hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i.e., CN), haloalkyl, aminoalkyl, hydroxyalkyl, cycloalkyl. Preferably, Q may be selected from substituted or unsubstituted C5-C7 cycloalkyl.
Preferably, W may be selected from -(CH2)c-napthtyl,-(CH2)c-phenyl, -(CH2)C- biphenyl, -(CH2)c-indolyl, -(CH2)c-benzothiazolyl, wherein c is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. More preferably, W may be selected from -(CH2)-napthtyl, -(CH2)- phenyl, -(CH2)-biphenyl, -(CH2)-indolyl or -(CH2)-benzothiazolyl.
Preferably, each X may be O.
Accordingly, a particularly preferred linker connecting the PSMA binding entity to the inventive conjugate may be characterized by the fol lowing Structural Formula (6a):
Figure imgf000036_0001
In the conjugates according to the present i nvention and characterized by any of the structural formulas presented herein, the substituents or groups identified by placeholders may be (where applicable) defined as follows. D may preferably be selected from 1 ,4,7, 10-tetraazacyclododecane-1 ,4,7, 1 0- tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine- N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2-(4,7- bis(carboxymethyl)-1 ,4,7-triazonan-1 -yl)pentanedioic acid (NODAGA), 2-(4,7, 1 0- tris(carboxymethyl)-1 ,4,7,1 0-tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-1 -[methyl(2- carboxyethyl)-phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 1 5-tetraazabicyclo[9,3, 1 .]pentadeca-1 (1 5),1 1 , 1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}-amino)-pentyl]-N-hydroxysuccinamide (DFO), and Diethylenetriamine- pentaacetic acid (DTPA), and derivatives thereof. More preferably, D may be selected from DOTA, NODAGA, or derivatives thereof.
X may preferably be each independently selected from O, N, S or P. More preferably, each X may be O. R1 and R2 may preferably be each independently selected from H, F, CI, Br, I, branched, unbranched or cyclic C1-G2 hydrocarbyl, C2-C12 alkenyl, C2-C12 alkylnyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, CONR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, or R1 and R2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic C1 -C10 hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optional ly substituted by up to 3 groups independently selected from F, CI, Br, I, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, and SR7 , =0, =S and =NH, wherein R6 and R7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl. More preferably, R1 may be H and R2 may be selected from halogen, preferably iodine or bromine, and C1-6 alkyl, preferably C1-3 alkyl, even more preferably methyl. Even more preferably, R1 may be H and R2 may be H or may be in the para position and selected from iodine, bromine and methyl. Y may preferably be selected from a single bond or a linear, branched or cyclic G-
C12 alkyl, optionally interrupted by up to two heteroatoms, optionally substituted by at least one halogen, branched, unbranched or cyclic G-Go hydrocarbyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, wherei n one or more of the non- adjacent CH2-groups may independently be replaced by -0-, -CO-, -CO-O-, -O-CO-, - NR6-, -NR6-CO- -CO-NR6-, -NR6-COO- -O-CO-NR6-, -NR6-CO-NR6— , -CH=CH- , - C≡C- -O-CO-O-, SR6-, SO3R6-, wherein R6 and R7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl. More preferably, Y may be may be a linear or branched, optionally substituted, G -G2 hydrocarbyl, more preferably a linear or branched, optionally substituted, G-G0 hydrocarbyl, even more preferably a linear or branched, optionally substituted, G-Q, hydrocarbyl, even more preferably a a linear or branched, optionally substituted, G-C3 hydrocarbyl. Most preferably, Y may be -(CH2)3-.
R3, R4 and R5 may preferably each be i ndependently selected from -COH, -CO2H, - SO2H, -SO3H, -SO4H, -PO2H, -PO3H, -PO4H2, -C(0)-(G -Go)alkyl, -C(0)-0(G-Go)alkyl, - C(0)-NHR8, or -C(0)-NR8R9* wherein R8 and R9 are each independently selected from H, bond, (C1 -C1 0)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(O)- (G-Go)alkylene, -(CH2)P-NH, -(CH2)P-(G -G0)alkyene, -(CH2)p-NH-C(0)-(CH2)q, -(CHrCH2)r NH-C(0)-(CH2)p, -(CH2)p-CO-COH, -(CH2)p-CO-C02H, -(CH2)p-C(0)NH-C[(CH2)q-COH]3, - C[(CH2)p-COH]3, -(CH2)p-C(0)NH-C[(CH2)q-C02H] 3, -C[(CH2)p-C02H]3 or -(CH2)P-(C5- Ci4)heteroaryl. More preferably, R3, R4 and R5 may be -C02H.
The spacer may preferably comprise at least one C-N bond. More preferably, the spacer may be characterized by Formula (3a), (3b) or (3c) as defined herein.
The linker may preferably be characterized by General Formula (6) as defined herein. More preferably, the linker may be characterized by Formula (6a) as defined herein. Q may preferably be selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl, preferably from substituted or unsubstituted C5-G 4 aryl, C5-C14 alkylaryl or C5-C14 cycloalkyl.
W may preferably be selected from -(CH2)c-aryl or -(CH2)c-heteroaryl, wherein c is preferably an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 .
A may preferably be an amino acid residue. More preferably, A may be selected from (D-) Aspartate, (D-)Glutamate or (L-Lysine). V may preferably be selected from a single bond, N, or an optionally substituted G-
Ci2 hydrocarbyl comprising up to 3 heteroatoms, wherein said heteroatom is preferably selected from N. n may preferably an integer selected from 1 , 2, 3, 4 or 5, preferably from 1 , 2 or 3, m may preferably be 0 or 1 . a, b, p, q, r, t may preferably each be independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0.
In accordance with the above, preferred conjugates according to the present invention may be characterized by General Formula (1 a):
Figure imgf000039_0001
d a) wherein
D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2 -(4,7,1 0-tris(carboxymethyl)-1 ,4,7, 1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-l -[methyl(2-carboxyethyl)- phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 1 5-tetraazabicyclo[9,3, 1 .]pentadeca-1 (1 5), 1 1 ,1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and
Diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,
X is each independently selected from O, N, S or P,
R1 and R2 are each independently selected from H, F, CI, Br, I, branched, unbranched or cyclic G-C12 hydrocarbyl, C2-C12 alkenyl, C2-C12 alkylnyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, CONR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, or R1 and R2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic G-Go hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optionally substituted by up to 3 groups independently selected from F, CI, Br, I, OR5, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, and SR7 , =0, =S and =NH,
Y is selected from a single bond or a linear, branched or cyclic, optional ly substituted G-G2 alkyl, optionally interrupted by up to two heteroatoms, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, wherein one or more of the non-adjacent CH2-groups may independently be replaced by -0-, -CO- -CO-0-, -0-CO-, -NR6-, -NR6-CO- -CO-NR6-, -NR6-COO- - O-CO-NR6-, -NR6-CO-NR6-, -CH=CH- , -C≡C- -0-CO-0-, SR6-, S03R6-,
R6 and R7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl,
R3, R4 and R5 are each independently selected from -COH, -C02H, -S02H, - SOsH, -SO4H, -PO2H, -PO3H, -P04H2, -C(0)-(G-Go)all<yl, -C(0)-0(G-Go)alkyl - C(0)-NHR8, or -C(0)-NR8R9' wherein R8 and R9 are each independently selected from H, bond, (G-Go)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(G-Go)alkylene, -(CH2)P-NH, -(CH2)p-(G-Go)alkyene, -(CH2)p-NH-C(0)-(CH2)q, -(CHrCH2)t-NH-C(0)-(CH2)p, -(CH2)p-CO-COH, -(CH2)p-CO-C02H, -(CH2)P-C(0)NH- C[(CH2)q-COH]3, -C[(CH2)p-COH]3, -(CH2)p-C(0)NH-C[(CH2)q-C02H]3, -C[(CH2)P- C02H]3 or -(CH2)p-(C5-Ci4)heteroaryl,
the spacer comprises at least one C-N bond,
the linker is characterized by General Formula (6) as defined above, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0, or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof. More preferably, the inventive conjugates may be characterized by General Formula (1 b)
Figure imgf000041_0001
(1 b) wherein
D is a chelator, preferably selected from 1 ,4,7,10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)benzyl]- ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane-1 ,4, 7- triacetic acid (NOTA), 2-(4,7-bis( carboxymethyl)-1 ,4,7-triazonan-1 -yl)pentanedioic acid (NODAGA), 2-(4,7,1 0-tris(carboxymethyl)-1 ,4,7, 1 0-tetraazacyclododecan-1 - yl)pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-1 -[methyl(2-carboxyethyl)phosphinic acid]-4,7- bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 1 5-tetraazabicyclo- [9,3, 1 .]pentadeca-1 (1 5),1 1 ,1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5- [Acetyl(hydroxy)amino] pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}-amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylenetriamine- pentaacetic acid (DTPA), or derivatives thereof,
Q is selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl,
W is selected from -(CH2)d-aryl or -(CH2)d-heteroaryl,
R1 and R2 are each independently selected from H, F, CI, Br, I, branched, linear or cyclic O-C12 hydrocarbyl optional ly comprising up to 2 heteroatoms and optional ly substituted by up to 3 groups independently selected from F, CI, Br, I, branched, unbranched or cyclic C1 -G2 hydrocarbyl, OR7, OCOR7, COOR7, CHO, COR7 CH2OR7, NR7R8, CH2NR7R8, and SR8 , =O, =S and =NH, wherein R7 and R8 are each independently selected from H or branched, unbranched or cyclic G -12 hydrocarbyl; preferably R1 and R2 are each independently selected from H, Br, I and linear G-C12 alky; R3, R4 and R5 are each independently selected from -COH, -C02H, -S02H, - S03H, -SO4H, -PO2H, -P03H, -P04H2/ -C(0)-(Ci -Cio)alkyl, -C(0)-0(Ci -Cio)alkyl, - C(0)-NHR8, or -C(0)-NR8R9' wherein R8 and R9 are each independently selected from H, bond, (Ci -Cio)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(Ci-C,o)alkylene, -(CH2)P-NH, -(CH2)P-(Ci -Ci0)alkyene, -(CH2)p-NH-C(0)-(CH2)q, -(CHrCH2)rNH-C(0)-(CH2)p, -(CH2)p-CO-COH, -(CH2)p-CO-C02H, -(CH2)P-C(0)NH- C[(CH2)q-COH]3, -C[(CH2)p-COH]3, -(CH2)p-C(0)NH-C[(CH2)q-C02H]3, -C[(CH2)P- C02H]3 or -(CH2)p-(C5-Ci4)heteroaryl,
a, b, d, p, q, r, s and t are each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0, and
the spacer comprises at least one C-N bond, or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof.
In preferred conjugates of the invention according to General Formula (1 b), any one of the following definitions, preferably at least two, more preferably at least three, more preferably at least four, or most preferably all of the following definitions may apply for "D", "Q", "W", "a", "b", "R1", "R2", R3", "R4" and/or "R5" :
D may be selected from any suitable chelator (e.g. as defined herein), more preferably D may be selected from from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof. Most preferably, D may be selected from DOTA, NODAGA, D03AP, D03APPrA or D03APABn.
Q may be selected from substituted or unsubstituted C5-C7 cycloalkyl. W may be selected from -(CH2)-napthtyl, -(CH2)-phenyl, -(CH2)-biphenyl, -(CH2)-indolyl or -(CH2)- benzothiazolyl, more preferably W may be -(CH2)-napthtyl. a, b may each independently be an integer selected from 0, 1 , 2, 3, 4, 5 or 6.
R1 and R2 may each independently be selected from H, iodine and G-C3 alkyl, and R3, R4 and R5 may each be C0 H. Such preferred conjugates may be characterized by General Formula (1 c):
Figure imgf000043_0001
(1 c) wherein
any one, preferably at least two, more preferably at least three, or most preferably all of the below definitions may apply for "D","a", "R1", and/or "R2":
D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof. Most preferably, D may be selected from DOTA, NODAGA, D03AP, D03APPrA or D03APAB ,
a may be an integer selected from 0, 1 , 2, 3, 4, 5 or 6,
R1 and R2 are each independently selected from H, iodine or G-G alkyl, and the spacer comprises at least one C-N bond, or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof.
In preferred conjugates of General Formula (1 c),
a may be 0, and
the spacer may be
Figure imgf000043_0002
wherein R10 and R11 may each be independently selected from H and branched, unbranched or cyclic G-G2 hydrocarbyl and wherein u may be an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0. In preferred conjugates of General Formula (1 a), the spacer is characterized by Formula (3a). Accordingly, such preferred conjugates may be characterized by General Formula (7a):
Figure imgf000044_0001
(7a) wherein
D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof. Most preferably, D may be selected from DOTA, NODAGA, D03AP, D03APPrA or D03 APABn,
R1 and R2 may each be independently selected from H, iodine or G-Cj alkyl, or pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof,
Specifically, preferred conjugates according to the invention may be characterized by Formula (7a)(i) or (7a)(ii):
Figure imgf000044_0002
(7a)(i) or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof
Conjugates characterized by Formula (7a)(i) are also referred to as "PSMA-06" herein.
Figure imgf000045_0001
(7a)(ii) or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof
Conjugates characterized by Formula (7a)(ii) are also referred to as "PSMA-03" herein.
In alternatively preferred conjugates of General Formula (1 c), the spacer comprises at least one amino acid residue, preferably selected from (D-/L-) aspartate, glutamate or lysine. Preferably, the spacer may comprise at least 1 , 2, 3, 4 or up to five 5 amino acids residue(s), preferably independently selected from (D-/L-) aspartate, glutamate or lysine ami no acid residues. Such conjugates may preferably comprise a spacer according to General Formula (3b) or (3c). Accordingly, such preferred conjugates may be characterized by General Formula (7b):
Figure imgf000046_0001
(7b) wherein
D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA,
TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof. Most preferably, D may be selected from DOTA, NODAGA, D03AP, D03APPrA or D03APABn,
R1 and R2 are each independently selected from H, iodine or C1-C3 alkyl,
A is an amino acid residue preferably selected from (D-)Aspartate, (D-)Glutamate or
(L-Lysine),
V is selected from a single bond, N, or an optionally substituted C1 -C12 hydrocarbyl comprising up to 3 heteroatoms, wherein said heteroatom is preferably selected from N,
n is an integer selected from 1 , 2, 3, 4 or 5, preferably from 1 , 2 or 3,
and a is an integer selected from 1 , 2, 3, 4, 5 or 6. or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof.
Figure imgf000047_0001
or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof. Conjugates characterized by Formula (7b)(i) are also referred to as PSMA-05 herein.
Figure imgf000048_0001
or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof. Conjugates characterized by Formula (7b)(ii) are also referred to as "PSMA-07" herein.
Figure imgf000048_0002
(7b)(iii) or pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof. Conjugates characterized by Formula (7b)(i i i) are also referred to as "PSMA-08" herei n.
Figure imgf000049_0001
(7b)(iv) or pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof Conjugates characterized by Formula (7b)(iv) are also referred to as "PSMA-04" herei n.
Pharmaceutically acceptable salts
The present i nvention further encompasses pharmaceutically acceptable salts of the conjugates described herei n.
The preparation of pharmaceutical compositions is wel l known to the person ski l led in the art. Pharmaceutical ly acceptable salts of the conjugates of the invention can be prepared by conventional procedures, such as by reacti ng any free base and/or acid of a conjugate accordi ng to the i nvention with at least a stoichiometric amount of the desi red salt- formi ng acid or base, respectively.
Pharmaceutical ly acceptable salts of the i nventive i nclude salts with i norganic cations such as sodium, potassium, calcium, magnesium, zi nc, and ammonium, and salts with organic bases. Suitable organic bases i nclude N-methyl-D-glucami ne, argmme, benzathi ne, diolamine, olamine, procaine and tromethamine. Pharmaceutically acceptable salts according to the invention also include salts derived from organic or inorganic acids. Suitable anions include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, maleate, mesylate, methylbromide, methylsulfate, napsylate, nitrate, oleate, pamoate, phosphate, polygalacturonate, stearate, succinate, sulfate, subsalicylate, tannate, tartrate, terephthalate, tosylate and triethiodide.
Complexed/Non-complexed forms
The present invention further encompasses the conjugates described herein, wherein the chelating agent D may be complexed with a metal ion (such as a radionuclide) or may not be complexed.
The term "radionuclide" (or "radioisotope") refers to isotopes of natural or artificial origin with an unstable neutron to proton ratio that disintegrates with the emission of corpuscular (i.e. protons (alpha-radiation) or electrons (beta-radiation) or electromagnetic radiation (gamma-radiation). In other words, radionuclides undergo radioactive decay, chelating agent D may be complexed with any knwon radionuclide. Said radionucl ide which may preferably be useful for cancer imaging or therapy. Such radionuclides include, without limitation, 94Tc, 99mTc, 90ln, 1 l 1 ln, 67Ga, 68Ga, 86Y, 90Y, 177Lu, 151Tb, 186Re, 188Re, 64Cu, 67Cu, 55Co, 57Co, 43Sc, 44Sc, 47Sc, 225Ac, 213Bi, 212Bi, 12Pb, 2 7Th, 153Sm, 166Ho, 152Gd, 153Gd, 157Gd, or 166Dy. The choice of suitable radionuclides may depend inter alia on the chemical structure and chelating capability of the chelating agent D, and the intended application of the resulting (complexed) conjugate (e.g. diagnostic vs. therapeutic). For instance, the beta-emitters such as 90Y, 131l, 161Tb and 177Lu may be used for concurrent systemic radionuclide therapy. Providi ng DOTA as a chelator may advantageously enable the use of either 68Ga, 43, 4' 7Sc, 177Lu, 16lTb, 225Ac, 213Bi, 212Bi, 212Pb as radionuclides. Esters and Prodrugs
The present invention further encompasses the inventive conjugates in their esterified form, in particular where free carboxylic acid groups are esterified. Such esterified compounds may be produg forms of the inventive conjugates. Suitable ester prodrugs include various alkyl esters, including saturated and unsaturated Cs-Cie fatty acids.
Enantiomers
The conjugates disclosed herein may exist in particular geometric or stereoisomeric forms. In addition, compounds may also be optically active. The inventive conjugates may also include cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. If, for instance, a particular enantiomer of a group or conjugate is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxi liary, where the resulting diastereomeric mixture is separated and the auxi liary group cleaved to provide the pure desired enantiomers. Alternatively, where the group or conjugate contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
A stereoisomer" is one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereo isomer of the compound and less than about 20% by weight of other stereo isomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 1 0% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereo isomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.
Radiolabeled complexes
According to a further aspect, the present invention relates to the use of the inventive conjugate for the preparation of radiolabeled complexes. Such radiolabeled complexes preferably comprise a conjugate according to the present invention, and a radionuclide. The chelating agent D preferably coordinates the radionuclide, forming a radiolabeled complex. Suitable radionuclides may be selected from theragnostic metal isotopes and comprise without limitation, 94Tc, 99mTc, 90ln, 111 In, 67Ga, 68Ga, 86Y, 90Y, 177Lu, 151Tb, 186Re, 188Re, 64Cu, 67Cu, 55Co, 57Co, 3Sc, 44Sc, 47Sc, 225Ac, 213Bi, 212Bi, 21 Pb, 2 7Th, 153Sm, 166Ho, 152Gd, 153Gd, 157Gd, or 166Dy.
According to a further aspect, the present invention further provides a complex comprising a radionuclide (preferably selected from the group above) and a conjugate according to the invention.
Pharmaceutical composition
According to a further aspect, the present invention provides a pharmaceutical composition comprising the inventive conjugate (including pharmaceutically acceptable salts, esters, solavtes or radiolabeled complexes as described herein), and a pharmaceutically acceptable carrier and/or excipient.
The term pharmaceutically acceptable" refers to a compound or agent that is compatible with the inventive conjugate and does not interfere with and/or substantially reduce its diagnostic or therapeutic activities. Pharmaceutically acceptable carriers preferably have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject to be treated.
Form u l ati on s, ca rri ers a n d exc i p i e nts
Pharmaceutical ly acceptable excipients can exhibit different functional roles and include, without limitation, di luents, fi llers, bulking agents, carriers, disintegrants, binders, lubricants, gl idants, coati ngs, solvents and co-solvents, bufferi ng agents, preservatives, adjuvants, anti-oxidants, wetti ng agents, anti-foaming agents, thickening agents, sweetening agents, flavouri ng agents and humectants. Suitable pharmaceutically acceptable excipients are typical ly chosen based on the formulation of the (pharmaceutical) composition.
For (pharmaceutical) compositions in liquid form, useful pharmaceutically acceptable excipients in general include solvents, di luents or carriers such as (pyrogen-free) water, (isotonic) saline solutions such phosphate or citrate buffered sal i ne, fixed oi ls, vegetable oi ls, such as, for example, groundnut oi l, cottonseed oi l, sesame oi l, ol ive oi l, corn oi l, ethanol, polyols (for example, glycerol, propylene glycol, polyetheylene glycol, and the l ike); lecithin; surfactants; preservatives such as benzyl alcohol, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the l i ke; isotonic agents such as sugars, polyalcohols such as manitol, sorbitol, or sodium chloride; alumi num monostearate or gelati n; antioxidants such as ascorbic acid or sodium bisulfite; chelati ng agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydroch loric acid or sodium hydroxide. Buffers may be hypertonic, isotonic or hypotonic with reference to the specific reference medi um, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherei n preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cel ls due to osmosis or other concentration effects. Reference media are e.g. l iquids occurri ng i n in vivo methods, such as blood, lymph, cytosolic l iquids, or other body liquids, or e.g. l iqu ids, which may be used as reference media i n in vitro methods, such as common buffers or l iquids. Such common buffers or l iquids are known to a ski l led person.
Liquid (pharmaceutical) compositions admi nistered via injection and i n particular via i .v. i njection should preferably be steri le and stable under the conditions of manufacture and storage. Such compositions are typical ly formulated as parenteral ly acceptable aqueous solutions that are pyrogen-free, have suitable pH, are isotonic and maintai n stabi l ity of the active ingredient(s). For liquid pharmaceutical compositions, suitable pharmaceutical ly acceptable excipients and carriers i nclude water, typical ly pyrogen-free water; isotonic sal ine or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions. Particularly for i njection of the i nventive (pharmaceutical) compositions, water or preferably a buffer, more preferably an aqueous buffer, may be used, which may contain a sodium salt, e.g. at least 50 mM of a sodium salt, a calcium salt, e.g. at least 0,01 mM of a calcium salt, and optional ly a potassium salt, e.g. at least 3 mM of a potassium salt.
The sodium, calcium and, optional ly, potassium salts may occur i n the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without bei ng l imited thereto, examples of sodium salts i nclude e.g. NaCI, Nal, NaBr, Na2C03, NaHCC , a2S04, examples of the optional potassium salts i nclude e.g. KG, Kl, KBr, K2CO3, KHCOs, K2SO4, and examples of calcium salts i nclude e.g. CaC , Cab, CaBr2, CaCCh, CaS04, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained i n the buffer.
Buffers suitable for i njection purposes as defined above, may contai n salts selected from sodium chloride (NaCI), calcium chloride (CaCI2) and optionally potassium chloride (KG), wherei n further anions may be present additional to the chlorides. CaCI2 can also be replaced by another salt like KG. Typical ly, the salts i n the injection buffer are present i n a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium ch loride (KG) and at least 0,01 mM calcium chloride (CaCI2). The i njection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i .e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherei n preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
For (pharmaceutical) compositions in (semi-)sol id form, suitable pharmaceutical ly acceptable excipients and carriers i nclude bi nders such as microcrystal l i ne cel lulose, gum tragacanth or gelatin; starch or lactose; sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cel lulose and its derivatives, such as, for example, sodium carboxymethylcel lulose, ethylcel lulose, cel lulose acetate; disintegrants such as algi nic acid; lubricants such as magnesium stearate; gl idants such as stearic acid, magnesium stearate; calcium sulphate, colloidal silicon dioxide and the like; sweetening agents such as sucrose or saccharin; and/or flavoring agents such as peppermint, methyl salicylate, or orange flavoring. Generally, (pharmaceutical) compositions for topical administration can be formulated as creams, ointments, gels, pastes or powders. (Pharmaceutical) compositions for oral administration can be formulated as tablets, capsules, liquids, powders or in a sustained release format. However, according to preferred embodiments, the inventive (pharmaceutical) composition is administered parenterally, in particular via intravenous or intratumoral injection, and is accordingly formulated in liquid or lyophi lized form for parenteral administration as discussed elsewhere herein. Parenteral formulations are typically stored in vials, IV bags, ampoules, cartridges, or pref i I led syringes and can be administered as injections, inhalants, or aerosols, with injections being preferred. The (pharmaceutical) composition may be provided in lyophilized form. Lyophi lized
(pharmaceutical) compositions are preferably reconstituted in a suitable buffer, advantageously based on an aqueous carrier, prior to administration.
The (pharmaceutical) composition preferably comprises a safe and effective amount of the inventive conjugate(s) or radiolabeled complexe(s).
As used herein,„safe and effective amount" means an amount of the agent(s) that is sufficient to allow for diagnosis and/or significantly induce a positive modification of the disease to be treated. At the same time, however, a„safe and effective amount" is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. A„safe and effective amount" will furthermore vary in connection with the particular condition to be diagnosed or treated and also with the age and physical condition of the patient to be treated, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutical ly acceptable excipient or carrier used, and similar factors. The inventive conjugates are also provided for use in the preparation of a medicament, preferably for treating cancer, in particular for treating and/or preventing prostate cancer, pancreatic cancer, renal cancer or bladder cancer. Kit
According to a further aspect, the present invention relates to a kit comprising the inventive conjugate(s) (including pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof) and/or a pharmaceutical composition(s) of the invention. Optionally, the kit may comprise at least one further agent as defi ned herein in the context of the pharmaceutical composition, including radionuclides, antimicrobial agents, solubilizing agents or the like.
The kit may be a kit of two or more parts comprising any of the components exemplified above in suitable containers. For example, each container may be in the form of vials, bottles, squeeze bottles, jars, sealed sleeves, envelopes or pouches, tubes or blister packages or any other suitable form, provided the container preferably prevents premature mixing of components. Each of the different components may be provided separately, or some of the different components may be provided together (i .e. in the same container).
A container may also be a compartment or a chamber within a vial, a tube, a jar, or an envelope, or a sleeve, or a blister package or a bottle, provided that the contents of one compartment are not able to associate physical ly with the contents of another compartment prior to their deliberate mixi ng by a pharmacist or physician.
The kit or kit-of-parts may furthermore contain technical i nstructions with information on the administration and dosage of any of its components. Therapeutic and diagnostic methods and uses
According to a further aspect, the present invention relates to the inventive conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical composition or kit for use in medicine and/or diagnostics. Preferably, said inventive conjugates, pharmaceutical compositions or kits are used for human medical purposes. Accordingly, the invention further encompasses these inventive conjugates, pharmaceutical composition or kit for use as a medicament.
The inventive conjugates are preferably capable of targeting prostate-specific membrane antigen (PSMA) in a selective manner. According to a specific aspect, the invention thus provides the inventive conjugates, pharmaceutical compositions or kits for use in a method of detecting the presence of cells and/or tissues expressing prostate-specific membrane antigen (PSMA). PSMA is in particular expressed on malignant cancer cells. As used herein, the term
„cancer" refers to a neoplasm characterized by the uncontrolled and usually rapid proliferation of cells that tend to invade surrounding tissue and to metastasize to distant body sites. The term encompasses benign and malignant neoplasms. Malignancy in cancers is typically characterized by anaplasia, invasiveness, and metastasis; whereas benign malignancies typically have none of those properties. The terms include neoplasms characterized by tumor growth as well as cancers of blood and lymphatic system.
Specifical ly, PSMA may be expressed, optionally in increased amounts, in prostate cancer cells, pancreatic cancer cells, renal cancer cells or bladder cancer cells.
According to a further specific aspect, the invention provides the inventive conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical composition or kit for use in a method of diagnosing, treating and/or preventing prostate cancer, pancreatic cancer, renal cancer or bladder cancer.
The term„diagnosis" or„diagnosing" refers to act of identifying a disease from its signs and symptoms and/or as in the present case the analysis of biological markers (such as genes or proteins) indicative of the disease. The term „treatment" or ^treating" of a disease includes preventing or protecting against the disease (that is, causing the clinical symptoms not to develop); inhibiting the disease (i.e., arresting or suppressing the development of clinical symptoms; and/or relieving the disease (i.e., causing the regression of clinical symptoms). As wi ll be appreciated, it is not always possible to distinguish between„preventing" and suppressing" a disease or disorder since the ultimate inductive event or events may be unknown or latent. Accordingly, the term „prophylaxis" will be understood to constitute a type of „treatment" that encompasses both „preventing" and suppressing." The term„treatment" thus includes„prophylaxis".
The term „subject", „patient" or individual" as used herein generally includes humans and non-human animals and preferably mammals (e.g., non-human primates, including marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, and baboons, macaques, chimpanzees, orangutans, gorillas; cows; horses; sheep; pigs; chicken; cats; dogs; mice; rat; rabbits; guinea pigs etc.), including chimeric and transgenic animals and disease models. In the context of the present invention, the term „subject" preferably refers a non-human primate or a human, most preferably a human.
The uses and methods described herein and relating to the diagnosis, treatment or prophylaxis of cancer, in particular prostate cancer, pancreatic cancer, renal cancer or bladder cancer, may preferably comprise the steps of (a) administering the inventive conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical composition or kit to a patient, and (b) obtaining a radiographic image from said patient.
The inventive conjugates, pharmaceutical compositions or kits are typical ly administered parenterally. Administration may preferably be accompl ished systemically, for instance by intravenous (i.v.), subcutaneous, intramuscular or intradermal injection. Alternatively, administration may be accomplished locally, for instance by intra-tumoral injection.
The inventive conjugates, pharmaceutical compositions or kits may be administered to a subject in need thereof several times a day, dai ly, every other day, sweekly, or monthly. Preferably, treatment, diagnosis or prophylaxis is effected with an effective dose of the inventive conjugates, pharmaceutical compositions or kits.
Effective doses of the inventive conjugates may be determined by routine experiments, e.g. by using animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Therapeutic efficacy and toxicity of inventive conjugates or radiolabeled complexes can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. The data obtained from the cell culture assays and animal studies can be used in determining a dose range for use in humans. The dose of said conjugates lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
For instance, therapeutical ly or diagnostically effective doses of the inventive conjugates may range from about 0.001 mg to 1 0 mg, preferably from about 0.01 mg to 5 mg, more preferably from about 0.1 mg to 2 mg per dosage unit or from about 0.01 nmol to 1 mmol per dosage unit, in particular from 1 nmol to 1 mmol per dosage unit, preferably from 1 micromol to 1 mmol per dosage unit. It is also envisaged that therapeutical ly or diagnostically effective doses of the inventive conjugates may range (per kg body weight) from about 0.01 mg/kg to 1 0 g/kg, preferably from about 0.05 mg/kg to 5 g/kg, more preferably from about 0.1 mg/kg to 2.5 g/kg. Advantageously, due to their favorable pharmacokinetic properties, the inventive conjugates may preferably be administered at lower doses than other PSMA ligands.
As established above, the inventive conjugates particularly lend themselves for theragnostic applications involving the targeting of PSMA-expressing cells. As used herein, the term „therangostic" includes„therapeutic-only", „diagnostic-only" and„thepeutic and diagnostic" applications. In a further aspect, the present invention relates to an in vitro method of detecting the presence of cells and/or tissues expressing prostate-specific membrane antigen (PSMA) comprising (a) contacting said PSMA-expressing cells and/or tissues with the inventive conjugates (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical compositions or kits and (b) applying detection means, optionally radiographic imaging, to detect said cells and/or tissues.
In the in vivo and in vitro uses and methods of the present invention, radiographic imaging may be accomplished using any means and methods known in the art. Preferably, radiographic imaging may involve positron emission tomography (PET) or single-photon emission computed tomography (SPECT). The targeted cells or tissues detected by radiographic imaging of the inventive conjugate may preferably comprise (optionally cancerous) prostate cells or tissues, (optionally cancerous) spleen cel ls or tissues, or (optionally cancerous) kidney cells or tissues.
In the in vivo and in vitro uses and methods of the present invention, the presence of PSMA-expressing cells or tissues may be indicative of a prostate tumor (cell), a metastasized prostate tumor (cel l), a renal tumor (cell), a pancreatic tumor (cell), a bladder tumor (cell), and combinations thereof. Hence, the inventive conjugates (including pharmaceutical ly acceptable salts, esters, solvates and radiolabeled complexes thereof), pharmaceutical compositions and kit may particularly be employed for diagnosis (and optional ly treatment) of prostate cancer, renal cancer, pracreatic cancer, or bladder cancer.
Description of the Figures
FIGURE 1 : Chromatograms of the HPLC-based quality control of (A) 177Lu-PSMA-ALB-01 , (B) 177Lu-PSMA-ALB-03, (C) 177Lu-PSMA-ALB-04, (D) 177Lu-PSMA-ALB-05, (E) 177Lu-PSMA-ALB- 06, (F) 177Lu-PSMA-ALB-07, and (G) 177Lu-PSMA-ALB-08 labeled at 50 MBq/nmol.
FIGURE 2: n-Octanol/PBS distribution coefficient of 177Lu-PSMA-ALB-01 (n=3), 177Lu-PSMA- ALB-03 (n=3), 177Lu-PSMA-ALB-04 (n=1 ), 17 Lu-PSMA-ALB-05 (n=l ), 177Lu-PSMA-ALB-06 (n=1 ), 177Lu-PSMA-ALB-07 (n=1 ), 177Lu-PSMA-ALB-08 (n=1 ) in comparison to the reference compound l 77Lu-PSMA-61 7 (n=3).
FIGURE 3: Data from ultrafiltration assays of 177Lu-PSMA-ALB-01 (n=2), 177Lu-PSMA-ALB-03 (n=2), 177Lu-PSMA-ALB-04 (n=1 ), 177Lu-PSMA-ALB-05 (n=1 ), 177Lu-PSMA-ALB-06 (n=2), 177Lu- PSMA-ALB-07 (n=2), 177Lu-PSMA-ALB-08 (n=2) in comparison to the reference compound 177Lu-PSMA-61 7 (n=2).
FIGURE 4: Uptake and internalization of l 77Lu-PSMA-ALB-01 (n=2), 177Lu-PSMA-ALB-03 (n=2), 177Lu-PSMA-ALB-04 (n=1 ), 177Lu-PSMA-ALB-05 (n=1 ), 177Lu-PSMA-ALB-06 (n=2), 177Lu- PSMA-ALB-07 (n=2), 177Lu-PSMA-ALB-08 (n=2) in comparison to the reference compound 17 Lu-PSMA-61 7 (n=3). (A&C) Data obtained in PSMApos PC-3 PIP cells. (B&D) Data obtained in PSMAneg PC-3 flu cells.
FIGURE 5: Biodistribution data of PC-3 PIP/flu tumor-bearing mice treated with 177Lu-PSMA- ALB-01 and 1 7Lu-PSMA-ALB-03 (A), 177Lu-PSMA-ALB-04 and 177Lu-PSMA-ALB-05 (B) and 177Lu-PSMA-ALB-06, 177Lu-PSMA-ALB-07 and 177Lu-PSMA-ALB-08 (C).
FIGURE 6: A conclusive selection of all (A) the tumor uptake, (B) the tumor/blood ratio, (C) the tumor/kidney ratio and (D) the tumor/liver ratio of 177Lu-PSMA-ALB-01 -08. Examples
Example 1 : Synthesis of PSMA ligands
All seven suggested PSMA ligands with a portable albumin-binding moiety were synthesized via a sol id-phase platform which was shown to be very useful for the development of above described albumin-affine PSMA ligands. A multistep synthesis (1 9 steps for PSMA-ALB-01 , 1 7 steps for PSMA-ALB-03, 20 steps for PSMA-ALB-04 and PSMA- ALB-05, 1 7 steps for PSMA-ALB-06, 23 steps for PSMA-ALB-07 and PSMA-ALB-08) provided these compounds in isolated overall yields of 26-49%. Crude products were purified by semi- preparative RP-HPLC assuring the final products with purities >98%. The characterization of above described compounds was performed by analytical RP-HPLC and MALDI-MS or ESI- MS, respectively. Analytical data are presented in Table 1 .
Table 1 : Analytical Data of PSMA-ALB-01 /03/04/05/06/07/08.
MW mlz>
Compound Code Chemical Formula
[g/mol] [min]
PSMA-ALB-01 C69H95I N14O20 1 567.50 1 568.59 8.1 5
PSMA-ALB-03 C65H92I N11 O18 1 442.41 1443.57 7.57
PSMA-ALB-04 C79H 116I N13O22 1 726.77 1 727.42 8.1 7
PSMA-ALB-05 C73H 102I N13O24 1 672.59 1 673.41 8.09
PSMA-ALB-06 C66H95N 11 O18 1 330.55 1 331 .47 7.24
PSMA-ALB-07 C77H107I N14O27 1 787.68 1 788.63 7.89
PSMA-ALB-08 C78H 110N 14O2 1 675.81 1 676.79 7.1 3
• Mass spectrometry of the unlabeled l igand detected as [M + HJ-; · Retention time of unlabeled l igand on analytical RP-HPLC. Analytical column (1 00 χ 4.6 mm) utilized Chromolith RP-1 8e stationary phase with mobile phases consisting of 0.1 % TFA in water (A) and ACN (B). For analytical runs, a linear gradient of solvent A (90-1 0% in 10 min) in solvent B at a flow rate of 1 mL/min was used.
The peptidomimetic pharmacophore for PSMA (L-Glu-NH-CO-NH-L-Lys binding entity; step 1 -6) was synthesized analogically as described by Eder et al. . Bioconjug. Chem. 201 2, 23: 688-697. The linker moiety (2-naphthyl-L-Ala-NH-CO-trans-CHX-N3 or 2- naphthyl-L-Ala-NH-CO-trans-CHx-Me-NH2; step 7-1 0) was prepared accordi ng to standard Fmoc (9-fluorenylmethyloxycarbonyl) protocol as previously introduced by Benesova et al. JNM 201 5, 56: 91 4-920. These two synthetic intermediate stages providing the PSMA ligand precursor were applied analogically for all four compounds (step 1 -8). However, the last building block of the linker area for PSMA-ALB-01 [trans-4-azidocyclohexanecarboxylic acid; step 9-1 0] was replaced for trans-4-(Fmoc-aminomethyl)cyclohexane-carboxylic acid (step 9-1 0) in case of PSMA-ALB-03/04/05/06/07/08.
PSMA -A L B-01
For synthesis of PSMA-ALB-01 , time-efficient „head-to-tai l" click coupling of the purified PSMA-precursor with the free azido group and the purified albumin-binding moiety [4-(p-iodophenyl)butyric acid-L-Lys] with propargyl-Gly (step 1 1 -1 7) was employed. After the efficient coupling of these two precursors via a triazole ring (step 1 8), an additional purification was performed to remove an excess of CuSCV5 H20. Final ly, PSMA-ALB-01 was obtained by the conjugation of the DOTA chelator in a form of its active ester (DOTA-NHS ester; step 1 9).
The Structural Formula of PSMA-ALB-01 is shown below:
Figure imgf000063_0001
(8)
PSMA -A L B-03
For the preparation of PSMA-ALB-03, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, DOTA tris(tBu)-ester conjugation, Alloc deprotection and 4-(p- iodophenyl)butyric acid conjugation fol lowed (step 1 1 -1 6). Final ly, PSMA-ALB-03 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H20 mixture (step 1 7).
The Structural Formula of PSMA-ALB-03 is shown below:
Figure imgf000064_0001
(PSMA-ALB-03)
PSMA -A L B-04
For the synthesis of PSMA-ALB-04, time-efficient„head-to-tail" coupling of the resin- coated PSMA-precursor with the DOTA-conjugated L-Lys and the purified albumin-binding moiety [4-(p-iodophenyl)butyric acid-L-Lys] through direct conjugation of two secondary amines (step 1 1 -1 8) was employed. After the efficient coupling of these two precursors using suberic acid bis(N-hydroxysuccinimide) ester (step 1 9), PSMA-ALB-04 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H20 mixture (step 20).
The Structural formula of PSMA-ALB-04 is shown below:
Figure imgf000065_0001
(PSMA-ALB-04)
PSMA -A L B-05
For the preparation of PSMA-ALB-05, straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupling to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, 4-(p-iodophenyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation followed (step 1 1 -1 9). PSMA-ALB-05 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H20 mixture (step 20).
The Structural Formula of PSMA-ALB-05 is shown below:
Figure imgf000065_0002
PSMA -A L B-06
For the synthesis of PSMA-ALB-06, straight one-way synthesis on the resi n support was employed. After the Fmoc-L-Lys(Alloc)-OH coupl i ng to PSMA-precursor, Fmoc deprotection, DOTA tris(tBu)-ester conjugation, Al loc deprotection and p-(tolyl)butyric acid conjugation followed (step 1 1 -1 6). Finally, PSMA-ALB-06 was obtained by agitation and subsequent cleavage from the resi n with TFA:TI PS:H20 mixture (step 1 7).
The Structural Formula of PSMA-ALB-06 is shown below:
Figure imgf000066_0001
(PSMA-ALB-06)
PSMA -A L B-07
For the preparation of PSMA-ALB-07, straight one-way synthesis on the resi n support was employed. After the Fmoc-L-Lys(Al loc)-OH coupl ing to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, 4-(p-iodophenyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation fol lowed (step 1 1 -22). PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resi n with TFA:TI PS:H20 mixture (step 23).
The Structural Formu la of PSMA-ALB-07 is shown below:
Figure imgf000067_0001
PSMA -A L B-08
For the preparation of PSMA-ALB-08 straight one-way synthesis on the resin support was employed. After the Fmoc-L-Lys(Alloc)-OH coupl ing to PSMA-precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, p-(tolyl)butyric acid conjugation, Alloc deprotection and DOTA tris(tBu)-ester conjugation followed (step 1 1 -22). PSMA-ALB-08 was obtained by agitation and subsequent cleavage from the resin with TFA:TIPS:H20 mixture (step 23).
The Structural Formula of PSMA-ALB-08 is shown below:
Figure imgf000068_0001
Example 2: Detailed synthesis of PSMA-ALB-03-08
Synthesis of the Glutamate-Urea-Lysine Bindi ng Entity
2-Chlorotrityl chloride resin {(2-CT-Resin; Merck; Catalog number 85501 70005), 0.30 mmol, substitution capacity 1 .63 mmol/g, 1 00-200 MESH, 1 % DVB, total swel ling volume in CH2CI2 >4.2 mL/g, [1 84 mg]} in 5mL syringe with the filter and combi stopper was first agitated in anhydrous dichloromethane (DCM) for 45 min.
The 2-CT-resin was then washed three times with anhydrous DCM and followed by reaction with 1 .2 equiv of Alloc (/V-allyloxycarbonyl) as well as Fmoc (N- fluorenylmethoxycarbonyl) protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.36 mmol, 452.50 g/mol, [1 63 mg], (1 )} and 4.8 equiv of N,N- di isopropylethylamine {(DIPEA), 1 .44 mmol, 129.24 g/mol, 0.742 g/ml, [251 pL]} in 3 mL of anhydrous DCM. The coupling of the first protected ami no acid on the resin (2) proceeded over the course of 1 6 h with the gentle agitation. The L-lysine-immobilized resin (2) was washed three times with DCM1 and three times with DCM2. Unreacted chlorotrityl groups remaining on the resin were washed five times with the mixture of DCM, methanol (MeOH), and DIPEA in a ratio of 1 7:2:1 (20 mL). Subsequently, the resin with Alloc and Fmoc protected L-lysine was washed three times with DCM1 , three times with DCM2, three times with Ν,Ν-dimethylformamide (DMFI ), and, finally, three times with DMF2. Selective removal of Fmoc-protecting group was realized by washing with the 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 get product (3). Alloc protected L-lysine was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , and, finally, three times with DCM2.
In the next step, 10 equiv of tBu protected L-glutamate hydrochloride {(H-Glu(OtBu)- O u · HCI; Merck; Catalog number 8540960005), 3.0 mmol, 295.8 g/mol, [887 mg], i} were used for the generation of the isocyanate of the glutamyl moiety iii. An appropriate amount of /Bu-protected L-glutamate was dissolved in 1 50 mL of DCM2 followed by, shortly afterwards, the addition of 3 mL of DIPEA. This solution was added dropwise over 4 h to a flask with 1 mmol of ice-cooled bis(trichloromethyl)carbonate {(BTC; Sigma; Catalog number 1 521 7-10G), 296.75 g/mol, [297 mg], ii} in 5 mL of dry DCM.
The L-lysine-immobilized resin with one free NH2-group (3) was added afterwards in one portion to the solution of the isocyanate of the glutamyl moiety iii and stirred for 1 6 h in order to obtain resin-immobilized bis(tBu)-Glu-urea-Lys(Alloc) (4).
The obtained product (4) coated on the resin was filtered off and washed three times with DCM1 and three times with DCM2. Cleavage of Alloc-protecting group was realized by reaction with 0.1 5 equiv of TPP Pd { [tetrakis(triphenylphosphine)palladium(0); Sigma; Catalog number 21 6666-1 G], 0.045 mmol, 1 1 55.56 g/mol, [1 05 mg]} in the presence of 1 5 equiv of morpholine {4.5 mmol, 87.1 2 g/mol, 0.999 g/mL, [392 pL]} in 3 mL of anhydrous DCM. The amount of Pd and morpholine was divided into 2 portions and reacted successively by shaking each for 1 h. The reaction was performed in the dark using aluminum foil.
The resin was then washed three times with DCM1 , three times with DCM2, three times with DMF1 , and, fi nal ly, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1 % DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(sodium diethyldithiocarbamate trihydrate; Sigma; Catalog number D3506-1 00G), 225.31 g/mol} in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 ml_ DMF2). The resin-immobilized and bis(tBu)-protected Glu-urea-Lys (5) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , three times with DCM2, and, finally, three times with diethylether (Et20) and dried under vacuum.
Such prepared Prostate-specific Membrane Antigen (PSMA) binding entity (5) was used for the next reaction in order to synthesize all seven compounds (PSMA-ALB- 01 /03/04/05/06/07/08).
The outline of the whole previous synthesis of the bis(tBu)-protected Glu-urea-Lys pharmacophore is summarized in Scheme 1 .
Scheme 1 : Synthesis of the Glutamate-Urea-Lysine Binding Entity for PSMA-ALB- 01 /03/04/05/06/
Figure imgf000071_0001
c
Figure imgf000071_0002
a) 2-CT-Resin in DCM and DIPEA; b) 50% piperidine in DMF; c) iii in DCM; d) TPP palladium in DCM and morpholine; e) 1% DIPEA in DMF, diethyldithiocarbamate in DMF
The resin-immobilized and bis(tBu)-protected binding entity (5) was first agitated anhydrous DCM for 45 min. Pre-swollen pharmacophore was washed three times wi DCM2, three times with DMF1 , and three times with DMF2.
Synthesis of the Linker area
Relative to the resin (0.1 mmol), 4 equiv of Fmoc protected 2-naphthyl-L-alanine {( Fmoc-2Nal-OH; Bachem; Catalog number B-21 00), 0.40 mmol, 437.50 g/mol, [1 75.0 mg]} corresponding to the first building block of the linker area were activated with 3.96 equiv of HBTU {(0-(benzotriazol-1 -yl)-N,N,^N'-tetramethyluronium hexafluorophosphate; Sigma; Catalog number 12804-25G-F), 0.39 mmol, 379.24 g/mol, [1 47.9 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [71 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)-protected pharmacophore (5) and agitated for 1 h.
Subsequently, the resin with bis(tBu)-protected Glu-urea-Lys and Fmoc protected 2- naphthyl-L-alanine (6) was washed three times with DMF1 and three times with DMF2. Selective removal of the Fmoc-protecting group from compound (6) was realized by washing with the 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 products (7).
In the next step, 4 equiv of the second building block which correspond to azidocyclohexanecarboxylic acid {(N3-1 ,4-trans-CHC-OH; Iris Biotech; Catalog number HAA2235.0001 ), 0.40 mmol, 1 69.1 8 g/mol, [67.7 mg]} for PSMA-ALB-01 or to Fmoc protected tranexamic acid {(trans-4-(Fmoc-aminomethyl)cyclohexane-carboxylic acid; Sigma; Catalog number 58446-5G-F), 0.40 mmol, 379.45 g/mol, [1 51 .8 mg]} for PSMA-ALB- 03/04/05/06/07/08 were activated with 3.96 equiv of HBTU {(Sigma; Catalog number 1 2804- 25G-F), 0.39 mmol, 379.24 g/mol, [147.9 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [71 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen compound (7) and agitated for 1 hour.
Subsequently, the resin with bis(tBu)-protected Glu-urea-Lys-2-naphthyl-L-alanine and azidocyclohexanecarboxylic acid (8A) was washed three times with DMF1 , three times with DMF2, three times with DCM1 , three times with DCM2, and, finally, three times with Et20 and dried under vacuum. Final PSMA-precursor (9A) was obtained by the agitation and subsequent cleavage from the resin within 2 h with the mixture consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H20 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in acetonitrile (ACN) and water in a ratio of 1 :1 and purified via RP- HPLC. Additionally, the resin with bis(tBu)-protected Glu-urea-Lys-2-naphthyl-L-alanine and Fmoc protected tranexamic acid (8B) was washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the compound (8B) was realized by washing with the 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 products (9B).
The outline of the whole previous synthesis of the linker area is summarized in Scheme 2.
Scheme 2: Synthesis of the Li nker Area, Precursor for PSMA-ALB-03/04/05/06/07/08.
Figure imgf000074_0001
(7) (8B) (9B) a) Fmoc-2-Nal-OH, HBTU, DMF, DIPEA; b) 50% piperidine, DMF; c) Fmoc-AMCH, HBTU in DMF, DIPEA; d) 50% piperidine, DMF Synthesis of PSMA-ALB-03
Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [1 81 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [1 49 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobi lized bis(tBu)-protected PSMA precursor (9B) and agitated for 1 h.
Selective removal of Fmoc-protecting group from the resulting compound (10B) was realized by washing with the 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 (1 1 B). The conjugation of the chelator to the resin-immobilized compound (1 1 B) was performed with 2 equiv of DOTA-tris(t-Bu)ester {([2 -(4,7, 10-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.20 mmol, 572.73 g/mol [1 1 5 mg]}. The chelator building block was activated with 1 .98 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.1 98 mmol, 379.24 g/mol, [75 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 1 B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation. The resulting compound (12B) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , and, finally, three times with DCM2.
Cleavage of Alloc-protecting group from the compound (12B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 21 6666-1 G), 0.03 mmol, 1 1 55.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.1 2 g/mol, 0.999 g/mL, [262 pL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foi l.
The resin was then washed three times with DCM1 , three times with DCM2, three times with DMFl , and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1 % DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (1 3 B) was then washed three times with DMF1 and three times with DMF2.
Finally, for the coupling of the albumin-binding moiety, 4 equiv of iodophenyl-butyric acid {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.1 0 g/mol, [1 1 6 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (1 3B) and agitated for 1 h. The resulting compound (14B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with Et20 and dried under vacuum.
The final compound PSMA-ALB-03 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H20 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
The outline of the above described synthesis is summarized in Scheme 3.
Scheme 3: Coupling of the Albumin Binder, DOTA chelator and PSMA Precursor
PSMA-ALB-03.
Figure imgf000077_0001
(14B) (PSMA-ALB-03) a) Fmoc-Lys(Alloc)-OH, HBTU, DMF, DIPEA; b) 50% piperidine, DMF; c) DOTA tris(tBu)ester, HBTU, DIPEA, DMF; d) Pd catalysator, morpholine,
DCM; e) iodophenyl butyric acid, HBTU, DMF, DIPEA; f) TFA, TIPS, H20 95:2.5:2.5; Synthesis of PSMA-ALB-04
Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Dde as well as Fmoc protected L-lysine {(Dde-Lys(Fmoc)-OH; Merck; Catalog number 8540000001 ), 0.40 mmol, 532.63 g/mol, [21 3 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobi lized bis(^Bu)- protected PSMA precursor (9B) and agitated for 1 h. The resulting compound (10B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 0B) was realized by washing with the 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 (1 1 B).
The conjugation of the chelator to the resin-immobilized compound (1 1 B) was performed with 3 equiv of DOTA-tris(f-Bu)ester {([2-(4,7,1 0-tris(2-(f-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.30 mmol, 572.73 g/mol [1 71 mg]}. The chelator building block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobi lized and the DMF pre-swollen compound (1 1 B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
The resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Dde-protecting group from the resulting compound (12 B) was realized by washing with the mixture of 2 % hydrazine in DMF twice for 5 min and then once again for 1 0 min in order to obtain the product (1 3B).
Relative to the resin-coated product (1 3 B), 2 equiv of disuccinimidyl suberate {([suberic acid bis(/V-hydroxysuccinimide ester)]; Sigma; 68528-80-3), 0.20 mmol, 368.34 g/mol, [74 mg]} was activated with 1 .98 equiv of HBTU {(Sigma; Catalog number 1 2804- 25C-F), 0.1 98 mmol, 379.24 g/mol, [73 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml_, [70 μΙ_]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swol len product (1 3B) and agitated for 1 h.
The resulting compound (1 4B) was then washed three times with DMF1 and three times with DMF2.
The outline of the above described synthesis is summarized in Scheme 4.
Figure imgf000080_0001
a) Dde-Lys(Fmoc)-OH, HBTU, DMF, DIPEA; b) 50% piperidine, DMF; c) DOTA tris(tBu)ester, HBTU, DIPEA, DMF; d) 2% hydrazine, DMF; e) disuccinimidyl suberate, DMF, DIPEA; The synthesis was accompanied by the parallel preparation of the albumin- binding precursor starting from the 2-chlorotrityl chloride resin {(2-CT-Resin; Merck; Catalog number 85501 70005), 0.20 mmol, substitution capacity 1 .63 mmol/g, 1 00-200 MESH, 1 % DVB, total swelling volume in CH2CI2 >4.2 mL/g, [1 23 mg]} in 5mL syringe with the filter and combi stopper which was first agitated in anhydrous dichloromethane (DCM) for 45 min.
The 2-CT resin was then washed three times with anhydrous DCM and followed by reaction with 1 .2 equiv of Dde as well as Fmoc protected L-lysine {(Dde-Lys(Fmoc)-OH; Bachem; Catalog number E-3385.0001 ), 0.24 mmol, 532.64 g/mol, [128 mg] (1 5 B)} and 4.8 equiv of DIPEA {0.96 mmol, 129.24 g/mol, 0.742 g/mL, [1 67 pL]} in 3 mL of anhydrous DCM.
The coupling of the first protected amino acid on the resin (1 6B) proceeded over the course of 1 6 h with gentle agitation.
The L-lysine-immobilized resin (1 6B) was washed three times with DCM1 and three times with DCM2. Unreacted chlorotrityl groups remaining on the resin were washed five times with the mixture of DCM, MeOH, and DIPEA in a ratio of 1 7:2:1 (20 mL).
Subsequently, the resin with Dde and Fmoc protected L-lysine was washed three times with DCM1 , three times with DCM2, three times with DMF1 , and, finally, three times with DMF2. Selective removal of Fmoc-protecting group was realized by washing with the 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 get product (1 7B).
Dde protected L-lysine was then washed three times with DMF1 and three times with DMF2, three times with DCM1 , three times with DCM2 and, finally, three times with Et20 and dried under vacuum.
Such prepared resin-coated Dde protected L-lysine (1 7B) was split into two portions and one of them was used for the next reaction. This resin-coated product was agitated in anhydrous DCM for 45 min and subsequently washed three times with DMF and three times with DMF2. Relative to the lysine-coated resin, 4 equiv of iodophenyl-butyric acid {([4-(/ iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μΙ_]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (17B) and agitated for 1 h.
The resin with Dde protected L-lysine and iodophenyl-butyric acid (18B) was washed three times with DMF1 and three times with DMF2. Selective removal of Dde-protecting group from the resulting compound (18B) was realized by washing with the mixture of 2 % hydrazine in DMF twice for 5 min and then once again for 10 min in order to obtain the product (19B). The albumin-targeting moiety (20B) was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of 5% TFA in DCM. The mixture of solvents from the product was evaporated, crude product dissolved in ACN and water in a ratio of 1 :1 and purified via RP-HPLC. The outline of the above described synthesis is summarized in Scheme 5.
Scheme 5: Coupling of the Albumin Binder for PSMA-ALB-04.
Figure imgf000083_0001
(20B) (19B) (18B) a) 2-CT-Resin in DCM and DIPEA; b) 50% piperidine in DWIF; c) iodophenylbutyric acid, HBTU in DMF and DIPEA; d) 2% hydrazine in DMF; e) 5% TFA in
DCM
Finally, the conjugation of 3 equiv of purified albumin-targeting moiety (20B) to the resin immobilized product (14B) was performed. Product (20B) was dissolved in dry DMF and 100 μΐ of DIPEA was added. Two min after the addition of DIPEA, the solution (20B) was added to the resin-immobilized and DMF pre-swollen product (14B) and agitated for 1 h.
The resulting compound (21 B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and, finally, three times with Et20 and dried under vacuum.
The final compound PSMA-ALB-04 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H20 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC.
The outline of the above described synthesis is summarized in Scheme 6. Scheme 6: Coupling of the Albumin Binder and DOTA-conjugated PSMA precursor for
Figure imgf000084_0001
(PSMA precursor) (Albumin precursor) (PSMA-ALB-04) a) DMF, DIPEA; b) TFA:TIPS:H20;
Synthesis of PSMA-ALB-05
Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Al loc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [1 81 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.396 mmol, 379.24 g/mol, [1 49 mg]} in the presence of 4 equiv of DI PEA (0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(fl3u)-protected PSMA precursor (9B) and agitated for 1 h. The resulting compound (1 0B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 0B) was realized by washing with the 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 (1 1 B).
Relative to the lysine-coated PSMA precursor (1 1 B), 3 equiv of Fmoc as well as /Bu protected D-aspartate {(Fmoc-D-Asp-Ort3u; Merck; Catalog number 8521440001 ), 0.30 mmol, 41 1 .45 g/mol, [1 23 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.297 mmol, 379.24 g/mol, [1 12 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(rt3u)-protected PSMA precursor (1 1 B) and agitated for 1 h.
The resulting compound (12 B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (12B) was realized by washing with the 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 (1 3B).
Relative to the lysine and aspartate-coated PSMA precursor (1 3B), 3 equiv of Fmoc as well as /Bu protected D-aspartate {(Fmoc-D-Asp-O/Bu; Merck; Catalog number 8521440001 ), 0.30 mmol, 41 1 .45 g/mol, [1 23 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(fl3u)-protected PSMA precursor (1 3B) and agitated for 1 h. The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the 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 (1 5B).
Relative to the resin-coated product (1 5B), 4 equiv of iodophenyl-butyric acid {([4- (yO-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.1 0 g mol, [1 1 6 mg] } was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [1 49 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DI PEA, the solution was added to the resin-immobi lized and DMF pre-swollen product (1 5B) and agitated for 1 h. The resulting compound (1 6B) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , and, finally, three times with DCM2.
Cleavage of Alloc-protecting group from the compound (1 6B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 21 6666-1 G), 0.03 mmol, 1 1 55.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/mL, [262 pL]} in 3 ml_ of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil. The resin was then washed three times with DCM1 , three times with DCM2, three times with DMF1 , and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1 % DIPEA in DMF (300 μΙ_ DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 mi n with a solution of cupral {(Sigma; Catalog number D3506-1 00G), 225.31 g/mol} in DMF2 at the concentration of 1 5 img/mL (450 mg cupral in 30 mL DMF2). The resulting compound (1 7B) was then washed three times with DMF1 and three times with DMF2.
The conjugation of the chelator to the resin-immobilized compound (1 7B) was performed with 3 equiv of DOTA-tris(t-Bu)ester {([2 -(4,7, 1 0-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.30 mmol, 572.73 g/mol [1 71 mg]}. The chelator building block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 7B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
Such product (1 8B) washed three times with DMF1 and three times with DMF2, three times with DCM1 , three times with DCM2 and, final ly, three times with Et20 and dried under vacuum.
The final compound PSMA-ALB-05 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H20 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in ratio of 1 :1 and purified via RP-HPLC.
The outline of the above described synthesis is summarized in two parts of Scheme 7.
87
Figure imgf000088_0001
Scheme 7 (continued): Coupling of the Albumin Binding-moiety, DOTA Chelator and PSMA Precursor for PSMA-ALB-05...continued
Figure imgf000089_0001
h) Pd catalysator, morpholine, DCM; i) DOTA tris(tBu)ester, HBTU, DIPEA, DMF; j) TFA:TIPS:H20 Relative to the lysine-coated PSMA precursor (9), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [1 81 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.396 mmol, 379.24 g/mol, [1 49 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(tBu)- protected PSMA precursor (9) and agitated for 1 h.
Selective removal of Fmoc-protecting group from the resulting compound (1 0) was realized by washing with the 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 (1 1 ).
The conjugation of the chelator to the resin-immobi lized compound (1 1 ) was performed with 2 equiv of DOTA-tris(t-Bu)ester {([2 -(4,7, 1 0-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7, 1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.20 mmol, 572.73 g/mol [1 1 5 mg]}. The chelator building block was activated with 1 .98 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.1 98 mmol, 379.24 g/mol, [75 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 1 ). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation. The resulting compound (12) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , and, finally, three times with DCM2. Cleavage of Alloc-protecting group from the compound (12) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 21 6666-1 G), 0.03 mmol, 1 1 55.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.1 2 g/mol, 0.999 g/mL, [262 pL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foi l.
The resin was then washed three times with DCM1 , three times with DCM2, three times with DMF1 , and, final ly, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1 % DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (1 3) was then washed three times with DMF1 and three times with DMF2.
Finally, for the coupling of the albumin-binding moiety, 4 equiv of tolyl-butyric acid {([4-(p-tolyl)butyric acid]; ABCR; AB1 1 921 2), 0.40 mmol, 1 78.23 g/mol, [71 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μί]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (1 3) and agitated for 1 h.
The resulting compound (1 4) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , three times with DCM2, and, finally, three times with Et20 and dried under vacuum.
The final compound PSMA-ALB-06 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H20 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1 :1 and purified via RP-HPLC.
The outline of the above described synthesis is summarized in Scheme 8.
Scheme 8: Coupling of the Albumin Binder, DOTA chelator and PSMA Precursor PSMA-ALB-06.
Figure imgf000092_0001
(14) (PSMA-ALB-06)
a) Fmoc-Lys(Alloc)-OH, HBTU, DMF, DIPEA; b) 50% piperidine, DMF; c) DOTA tris(tBu)ester, HBTU, DIPEA, DMF; d) Pd catalysator, morpholine,
DCM; e) tolyl butyric acid, HBTU, DMF, DIPEA; f) TFA, TIPS, H20 95:2.5:2.5; Synthesis of PSMA-ALB-07
Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [1 81 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swol len immobilized bis(rt3u)-protected PSMA precursor (9B) and agitated for 1 h. The resulting compound (1 0B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 0B) was realized by washing with the 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 (1 1 B).
Relative to the lysine-coated PSMA precursor (1 1 B), 3 equiv of Fmoc as well as Bu protected D-aspartate {(Fmoc-D-Asp-0?Bu; Merck; Catalog number 8521 440001 ), 0.30 mmol, 41 1 .45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [1 12 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis( Bu)-protected PSMA precursor (1 1 B) and agitated for 1 h.
The resulting compound (1 2 B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 2 B) was realized by washing with the 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 (1 3B). Relative to the lysine and aspartate-coated PSMA precursor (1 3B), 3 equiv of Fmoc as well as iBu protected D-aspartate {(Fmoc-D-Asp-Ort3u; Merck; Catalog number 8521440001 ), 0.30 mmol, 41 1 .45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.297 mmol, 379.24 g/mol, [1 12 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(/Bu)-protected PSMA precursor (13B) and agitated for 1 h. The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the 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 (15B).
Relative to the lysine and two aspartates-coated PSMA precursor (15B), 3 equiv of Fmoc as well as (Bu protected D-aspartate {(Fmoc-D-Asp-O/Bu; Merck; Catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μΙ_]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(/Bu)-protected PSMA precursor (15B) and agitated for 1 h.
The resulting compound (16B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the 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 (17B). Relative to the resin-coated product (17B), 4 equiv of iodophenyl-butyric acid {([4-
(/ iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swollen product (17B) and agitated for 1 h. The resulting compound (1 8B) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , and, finally, three times with DCM2.
Cleavage of Al loc-protecting group from the compound (1 8B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 21 6666-1 G), 0.03 mmol, 1 1 55.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.1 2 g/mol, 0.999 g/mL, [262 pL]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil. The resin was then washed three times with DCM1 , three times with DCM2, three times with DMF1 , and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1 % DIPEA in DMF (300 μΙ_ DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-1 00G), 225.31 g/mol} in DMF2 at the concentration of 1 5 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (1 9B) was then washed three times with DMF1 and three times with DMF2.
The conjugation of the chelator to the resin-immobilized compound (1 9B) was performed with 3 equiv of DOTA-tris(t-Bu)ester {([2 -(4,7, 1 0-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,1 0-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 1 37076-54- 1 ), 0.30 mmol, 572.73 g/mol [1 71 mg]}. The chelator bui lding block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL] } in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (1 7B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
Such product (20B) washed three times with DMF1 and three times with DMF2, three times with DCM1 , three times with DCM2 and, finally, three times with Et20 and dried under vacuum.
The final compound PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in ratio of 1:1 and purified via RP-HPLC.
The outline of the above described synthesis is summarized in two parts of Scheme 9.
Scheme 9: Coupling of the Albumin Binding-moiety, DOTA Chelator and PSMA Precursor for PSMA-ALB-07.
Figure imgf000097_0001
Scheme 9 (continued): Coupling of the Albumin Binding-moiety, DOTA Chelator and PSMA Precursor for PSMA-ALB-07.
Figure imgf000098_0001
Figure imgf000098_0002
Synthesis of PSMA-ALB-08
Relative to the lysine-coated PSMA precursor (9B), 4 equiv of Fmoc as well as Al loc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; Catalog number 8521 240005), 0.40 mmol, 452.50 g/mol, [1 81 mg]} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [1 49 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swol len immobilized bis(rt3u)-protected PSMA precursor (9B) and agitated for 1 h. The resulting compound (1 0B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 0B) was realized by washing with the mixture of DMF and piperidine i n a ratio of 1 :1 once for 2 min and then once again for 5 min in order to obtain the product (1 1 B).
Relative to the lysine-coated PSMA precursor (1 1 B), 3 equiv of Fmoc as well as /Bu protected D-aspartate {(Fmoc-D-Asp-O/Bu; Merck; Catalog number 8521440001 ), 0.30 mmol, 41 1 .45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobilized bis(/Bu)-protected PSMA precursor (1 1 B) and agitated for 1 h.
The resulting compound (1 2 B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 2 B) was realized by washing with the 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 (1 3 B). Relative to the lysine and aspartate-coated PSMA precursor (1 3B), 3 equiv of Fmoc as well as /Bu protected D-aspartate {(Fmoc-D-Asp-Ort3u; Merck; Catalog number 8521440001 ), 0.30 mmol, 41 1 .45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.297 mmol, 379.24 g/mol, [1 1 2 mg]} i n the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μί]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swol len immobi lized bis(/Bu)-protected PSMA precursor (1 3B) and agitated for 1 h. The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (1 4B) was realized by washing with the 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 (1 5B).
Relative to the lysine and two aspartates-coated PSMA precursor (1 5B), 3 equiv of Fmoc as wel l as rt3u protected D-aspartate {(Fmoc-D-Asp-Ort3u; Merck; Catalog number 8521440001 ), 0.30 mmol, 41 1 .45 g/mol, [123 mg]} was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 1 2804-25G-F), 0.297 mmol, 379.24 g/mol, [1 12 mg] } in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 μΙ_]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the DMF pre-swollen immobi lized bis(?B unprotected PSMA precursor (1 5B) and agitated for 1 h.
The resulting compound (1 6B) was then washed three times with DMF1 and three times with DMF2. Selective removal of Fmoc-protecting group from the resulting compound (14B) was realized by washing with the 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 (1 7B). Relative to the resin-coated product (1 7B), 4 equiv of tolyl-butyric acid (0.40 mmol} was activated with 3.96 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [1 49 mg] } in the presence of 4 equiv of DIPEA {0.40 mmol, 1 29.24 g/mol, 0.742 g/mL, [70 μί]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin-immobilized and DMF pre-swol len product (1 7B) and agitated for 1 h.
The resulting compound (1 8B) was then washed three times with DMF1 , three times with DMF2, three times with DCM1 , and, finally, three times with DCM2. Cleavage of Alloc-protecting group from the compound (18B) was realized by reaction with 0.03 equiv of TPP Pd {(Sigma; Catalog number 216666-1 G), 0.03 mmol, 1155.56 g/mol, [35 mg]} in the presence of 30 equiv of morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/mL, [262 μΙ_]} in 3 mL of anhydrous DCM. The reaction was performed for 2 hours in the dark using aluminum foil.
The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and, finally, three times with DMF2. To remove residuals of the palladium, the resin was additionally washed ten times with 1% DIPEA in DMF (300 pL DIPEA in 30 mL DMF2) and subsequently washed ten times for 5 min with a solution of cupral {(Sigma; Catalog number D3506-100G), 225.31 g/mol} in DMF2 at the concentration of 15 mg/mL (450 mg cupral in 30 mL DMF2). The resulting compound (19B) was then washed three times with DMF1 and three times with DMF2.
The conjugation of the chelator to the resin-immobilized compound (19B) was performed with 3 equiv of DOTA-tris(t-Bu)ester {([2 -(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)- 1 ,4,7,10-tetraazacyclododecan-1 -yl)acetic acid]; CheMatech; Catalog number 137076-54- 1 ), 0.30 mmol, 572.73 g/mol [171 mg]}. The chelator building block was activated with 2.97 equiv of HBTU {(Sigma; Catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv of DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/mL, [70 pL]} in anhydrous DMF. Two min after the addition of DIPEA, the solution was added to the resin- immobilized and the DMF pre-swollen compound (17B). The coupling of the DOTA chelator proceeded over the course of 2 h with gentle agitation.
Such product (20B) washed three times with DMF1 and three times with DMF2, three times with DCM1, three times with DCM2 and, finally, three times with Et20 and dried under vacuum. The final compound PSMA-ALB-07 was obtained by agitation and subsequent cleavage from the resin within 2 h with a mixture consisting of TFA, TIPS and H20 in a ratio of 95:2.5:2.5. TFA was evaporated, crude product dissolved in ACN and water in a ratio of 1:1 and purified via RP-HPLC. The outline of the above described synthesis is summarized in two parts of Scheme 10.
Figure imgf000103_0001
f) Ġ Scheme 10 (continued): Coupling of the Albumin Binding-moiety, DOTA Chelator a PSMA Precursor for PSMA-ALB-08.
Figure imgf000104_0001
(PSMA-ALB-08) 1 '
h) Pd catalysator, morpholine, DCWl; i) DOTA tris(tBu)ester, HBTU, DIPEA, DMF; j) TFA:TIPS:H20 Example 3: 177Lu-LabeIing of PSMA ligands and In Vitro Evaluation
Introduction
In vitro studies were conducted with 177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08. This included the preliminary evaluation of labeling efficiencies, n-octanol/PBS distribution coefficients and serum protein binding studies. Furthermore, uptake and internalization experiments were performed using the PSMA-transfected PSMApos PC-3 PIP cell line (positive control) and the mock-transfected PSMAneg PC-3 flu cell line (negative control).
Materials & Methods PS MA- L i ga n ds a n d Ra d i o n u c l i d es
The PSMA-ligands 177Lu-PSMA-ALB-01 /-03/-04/-05/-06/-07/-08 were synthesized as described above (see Examples 1 and 2). The reference compound (PSMA-61 7) was purchased from Advanced Biochemical Compounds (ABX GmbH, Radeberg, Germany). No-carrier added 177Lu in 0.05 M HCI was provided by Isotope Technologies Garching (ITG GmbH, Germany).
Rad i o l a be l i ng
The stock solution of PSMA-61 7 was prepared by di lution in MilliQ water to a final concentration of 1 mM. l 77Lu-PSMA-ALB-01 /-03/-04/-05/-06/-07/-08 were diluted in Mil liQ water/DMSO to obtain a final concentration of 1 mM. All compounds were labeled with 177Lu in a 1 :5 mixture of sodium acetate (0.5 M, pH 8) and HCI (0.05 M, pH ~1 ) at pH 3.5-4.5. The compounds were labeled with 177Lu at specific activities between 5-50 MBq/nmol, depending on the experimental conditions. The reaction mixture was incubated for 1 5 min at 95°C, followed by a quality control using high-performance liquid chromatography with a C-1 8 reversed-phase column (XterraTM MS, C1 8, 5 μιη, 1 50x4.6 mm; Waters). The mobi le phase consisted of MilliQ water containing 0.1 % trifluoracetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% B to 20% A and 80% B over a period of 1 5 min at a flow rate of 1 .0 mL/min. The radioligands were diluted in MilliQ water containing Na-DTPA (50 μΜ (micromolar)) prior to injection into HPLC. Determination of the n-Octanol/PBS Distribution Coefficient
177Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 and PSMA-617 were labeled with 177Lu at a specific activity of 50 MBq/nmol. The radioligand (0.5 MBq; 10 pmol, 25 μ[_) was then added to a reagent tube containing 1475 μΐ of PBS pH 7.4 and 500 μΙ_ of n-octanol. The vials were vortexed vigorously followed by a centrifugation step for phase separation. Finally, the radioactivity in a defined volume of PBS and n-octanol was measured in a gamma-counter (Perkin Elmer, Wallac Wizard 1480) to calculate the distribution coefficients, expressed as the logarithm of the ratio of counts per minute (cpm) measured in the n-octanol phase to the cpm measure in the PBS phase. Determination of Albumin Binding of the PSMA Ligands Using a Filter Assay
Plasma binding of l77Lu-PSMA-ALB-01/-03/-04/-05/-06/-07/-08 and l77Lu-PSMA-617 was determined using an ultrafiltration assay.
Therefore, the compounds were labeled with '77Lu at a specific activity of 50 MBq/nmol and incubated in human plasma samples or PBS at room temperature. The free and plasma-bound fractions were separated using a centrifree ultrafiltration device (4104 centrifugal filter units [Millipore]; 30000 Da nominal molecular weight limit, methylcellulose micropartition membranes). The incubated solution was loaded to the ultrafiltration device and centrifuged at 2500 rpm for 40 min at 20°C. Samples from the filtrate were taken an analyzed for radioactivity in a gamma-counter. The amount of plasma-bound compound was calculated as the fraction of radioactivity measured in the filtrate relative to the corresponding loading solution (set to 100%).
Cell Internalization Studies
Cell uptake and internalization experiments were performed with 177Lu-PSMA-ALB- 01/-03/-04/-05/-06/-07/-08 and the reference compound l77Lu-PSMA-617 using the PSMA- transfected PSMAP°s PC-3 PIP and mock-transfected PSMANF¾ PC-3 flu cells in order to investigate the specificity of the novel compounds. Cells were grown in RPMI cell culture medium supplemented with 1 0% fetal calf serum, L-glutamine, antibiotics and puromycin (2 μg/mL) at 37°C and 5% C02 (standard conditions). Routine cell culture was performed twice a week using PBS/EDTA (2 mM) for washing the cells and trypsin for detachment of the cells. The cells were seeded in 12-well plates (~3 x 1 0s cel ls in 2 mL RPMI medium/well) allowing adhesion and growth overnight at standard conditions. The supernatant was removed and the cel ls washed with PBS pH 7.4 prior to the addition of RPMI medium without supplements (975 pL/well). The compounds were labeled with 177Lu at a specific activity of 5 MBq/nmol and diluted to 1 .5 MBq/mL in 0.05% bovine serum albumin (BSA)/0.9% NaCI solution to prevent adherence to plastic vessels. The cells were incubated with 25 μΙ_ (-37.5 kBq)/well radiolabeled PSMA ligands at standard conditions for 2 h and 4 h, respectively. After incubation, the cells were washed three times with ice-cold PBS and the total uptake of the radioligands was determined (PSMA- bound fraction on the surface and internalized fraction). The fraction of internalized radioligand was evaluated in cells washed with ice-cold PBS, followed by a 1 0 min incubation with stripping buffer (0.05 M glycine stripping buffer in 1 00 mM NaCI, pH 2.8) and an additional washing step with ice-cold PBS. Cell samples were lysed by addition of NaOH (1 M, 1 mL) to each well. The samples of the cell suspensions were measured in a γ- counter (Perkin Elmer, Wal lac Wizard 1 480). After homogenization of the cell suspensions, the protein concentration was determined for each sample using a Micro BCA Protein Assay kit (Pierce, Therma Scientific). The results were expressed as percentage of total added radioactivity per 1 50 g/mL protein.
Results
La be l i n g Eff i c i en cy PSMA-ALB-01 and -03 were successfully labeled with 177Lu at specific activities up to
1 00 MBq/nmol and excellent radiochemical yields of >98%. PSMA-ALB-04, -05, -06, -07 and -08 were labeled with 177Lu in preliminary tests at specific activities up to 50 MBq/nmol and excellent radiochemical yields of >97%. The specific activity used for the experiments (if not otherwise stated) was 50 MBq/nmol. The radiochemical purity of compounds used for in vitro and in vivo studies was always >97% (Fig.1 ). n-Octanol/PBS Distribution Coefficient l77Lu-PSMA-ALB-01, -03, -04 and -06 showed similar n-octanol/PBS distribution coefficients (LogD value), while the coefficients of 177Lu-PSMA-ALB-05, -07 and -08 indicated slightly more hydrophilic compounds. In general, the data showed that the introduction of an albumin-binding entity reduces the hydrophilicity as compared to the reference compound 177Lu-PSMA-617, however, all compounds are still hydrophilic with logD values > 2.7 (Fig 2).
Albumin-Binding Properties
The ultrafiltration experiments of 177Lu-PSMA-ALB-01 , -03, -04, -05, -06 and -07 revealed high serum protein binding capacities as >94% of the compound did not penetrate the filter when incubated in human plasma. The easy possibility of filtrating the compounds was demonstrating when incubating the compound sin PBS where proteins are not present (Fig.3). All newly designed compounds revealed increased serum protein binding capacity as compared to 177Lu-PSMA-617, which showed an albumin-bound fraction of only about 44% (Fig.3) Internalization
Cell uptake and internalization of PSMA ligands 177Lu-PSMA-ALB-01 , -03, -04, -05, - 06, -07 and -08 were investigated and compared to the reference compound 177Lu-PSMA-617 using PC-3 PIP/flu cells (Fig.4). The uptake of all compounds into PC-3 PIP cells (PSMAP°S) was comparable to 177Lu-PSMA-617 at 2 h or 4 h, respectively. Interestingly, the internalized fraction of the PSMA ligands was higher than for 177Lu-PSMA-617 at the 2h and 4 h time- point. The internalization rate of 177Lu-PSMA-ALB-06 and 177Lu-PSMA-ALB-08 was still comparable to 17 Lu-PSMA-617. The uptake of all radioligands in PC-3 flu cells (PSMAne¾ was <0.5%, which proved a highly PSMA-specific uptake/internalization of all compounds. Example 4: In Vivo Evaluation of PSMA ligands using PC-3 PIP/flu Tumor- Bearing Mice
Introduction
177Lu-PSMA-ALB-01 , -03, -04, -05, -06, -07 and -08 were characterized in vivo. Therefore, immunodeficient Balb/c nude mice were inoculated with PSMApos PC-3 PIP and PSMAneg PC-3 flu cells. After intravenous (i.v.) application of the ligands, extensive biodistribution and SPECT/CT studies were performed. Tumor uptake, tumor/blood ratio, tumor/kidney ratio and tumor/liver ratio of 177Lu-PSMA-ALB-01 -08 are summarized in Figure 6.
Materials & Methods
Tu mor Mo u se Mode l
Mice were obtained from Charles River Laboratories, Sulzfeld, Germany, at the age of 5-6 weeks. Female, athymic nude Balb/c mice were subcutaneously inoculated with PC-3 PIP cells (6 x 106 cells in 1 00 μΐ Hank's balanced salt solution (HBSS) with Ca2+/Mg2+) on the right shoulder and with PC-3 flu cells (5 x 1 06 cells in 1 00 μί HBSS Ca2+ Mg2+) on the left shoulder. Two weeks later, the tumors reached a size of about 200-300 mm3 suitable for the performance of the biodistribution and imaging studies.
B i od i stri b uti o n Stu d i es Biodistribution studies were performed using PC-3 PIP/flu tumor-bearing mice, which were inoculated with tumor cells two weeks prior to injection of PSMA ligands. The radioligands were di luted in 0.9% NaCI and i .v. injected in a volume of 1 00-200 μί. Mice were euthanized at different time points after injection (p.i.) of the radioligands. Selected tissues and organs were collected, weighed and measured using a gamma-counter. The results were decay-corrected and listed as a percentage of the injected activity per gram of tissue mass (% lA/g). Results
B i od i stri b uti o n of 1 77 L u - PSMA-A L B -01 , 1 77 L u - PS MA-A L B -03
The tissue distribution of 177Lu-PSMA-ALB-01 and 177Lu-PSMA-ALB-03 was investigated over a period of eight days. Compounds 177Lu-PSMA-ALB-01 and 177Lu-PSMA- ALB-03 showed highly similar tissue distribution profiles (Fig. 5A).
High radioactivity levels could be observed in the blood pool already at early time points and were cleared slowly but steadi ly over time. The uptake of both radioligands in the PSMApos PC-3 PIP tumors was increasing until it reached a plateau and did not drop substantially unti l the end of the study. The uptake in PC-3 flu tumors was clearly below blood levels, indicating highly PSMA-specific binding and uptake in vivo (Fig. 5A). Biodistribution data for 177Lu-PSMA-ALB-01 and -03 are shown in Table 2 and 3 below.
Table 2: Biodistribution of 177Lu-PSMA-ALB-01 in PC-3 PIP/flu Tumor-Bearing Mice
1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i.
Blood 29.7 ± 4.49 25.6 + 1 .53 21 .0 + 2.86 14.2 + 1.40 12.0 ± 2.18 6.68 ± 0.85
Heart 10.1 + 1.10 8.71 + 0.50 7.16 + 1.23 5.93 ± 0.65 4.42 + 0.81 2.70 + 0.37
Lung 16.6 ± 2.78 14.1 + 0.99 1 1 .6 + 0.83 8.62 ± 1.47 7.67 + 0.83 5.07 + 0.66
Spleen 5.27 + 1.64 5.34 ± 0.90 4.05 ± 0.69 3.60 + 0.43 4.62 ± 1.12 3.12 ± .015
Kidneys 19.4 + 4.82 24.6 ± 0.38 22.6 ± 2.38 22.7 + 2.18 25.2 ± 4.15 13.0 ± 1.30
Stomach 3.29 + 1.75 3.30 ± 0.05 2.45 ± 0.43 1.39 + 0.07 1 .49 ± 0.47 0.81 ± 0.04
Intestines 4.15 ± 1.40 4.17 ± 0.70 2.44 + 0.17 2.12 ± 0.20 1 .84 + 0.54 1 .05 + 0.17
Liver 5.76 ± 1.21 5.92 ± 0.07 5.31 + 1 .23 2.92 + 0.67 3.03 + 0.63 1 .88 + 0.36
Salivary glands 5.52 + 1 .08 5.20 + 0.73 4.45 + 0.56 3.38 + 0.32 3.96 ± 0.98 2.22 ± 0.38
Muscle 2.22 ± 0.88 2.06 + 0.80 1 .63 + 0.27 1 .34 ± 0.14 1 .35 ± 0.46 0.82 + 0.12
Bone 3.15 ± 0.47 3.01 ± 0.09 2.54 + 0.26 1.58 + 0.06 1 .64 ± 0.34 1 .07 + 0.22
PC-3 PIP Tumor 8.98 + 2.77 20.4 ± 0.39 25.5 ± 2.02 38.2 + 2.59 65.6 + 1 .84 62.3 ± 3.56
PC-3 flu Tumor 3.64 + 2.30 5.03 + 1 .61 4.01 + 0.79 3.95 ± 0.82 4.64 ± 1 .84 2.76 + 0.23
Tumor-to-blood 0.30 + 0.06 0.80 ± 0.03 1 .22 ± 0.08 2.71 + 0.39 5.54 ± 0.76 9.39 + 0.65
Tumor-to-liver 1 .56 + 0.30 3.45 ± 0.03 4.97 ± 1.21 13.5 ± 2.79 22.2 + 4.1 1 33.7 + 4.33
Tumor-to-kidney 0.46 + 0.04 0.83 + 0.03 1.13 + 0.07 1 .69 ± 0.07 2.64 ± 0.40 4.80 + 0.20
144 h .p.i. 192 h .p.i.
Blood 5.78 + 0.90 5.21 + 1.37
Heart 2.23 ± 0.29 2.12 ± 0.68
Lung 4.90 + 0.58 4.12 + 0.96
Spleen 4.32 + 0.57 4.09 ± 1.12 Kidneys 10.2 + 3.41 7.56 + 1.44
Stomach 0.84 + 0.13 0.73 + 0.12
Intestines 1.07 ± 0.13 1.02 ± 0.25
Liver 1.56 ± 0.16 1.51 ± 0.37
Salivary glands 1.68 + 0.53 1.78 + 0.34
Muscle 0.66 ± 0.15 0.64 ± 0.13
Bone 0.99 + 0.20 0.86 + 0.17
PC-3 PIP Tumor 78.4 + 8.57 75.6 + 22.0
PC-3 flu Tumor 2.82 + 0.24 2.73 + 0.84
Tumor-to-blood 13.8 + 2.38 14.5 + 1.84
Tumor-to-liver 50.5 ± 6.28 50.2 ± 7.38
Tumor-to-kidney 8.05 + 1.77 9.87 + 1.05
Table 3: Biodistribution of 177Lu-PSMA-ALB-03 in PC-3 PIP/flu Tumor-Bearing Mice
1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i.
Blood 27.4 ± 3.04 24.3 ± 3.60 23.5 ± 0.74 17.3 ± 1.38 12.5 + 3.78 7.37 + 0.64
Heart 9.64 ± 1.21 8.54 ± 1.18 8.12 ± 0.46 6.60 + 1.01 4.40 + 1.18 3.15 ± 0.28
14.21 ± 12 21 ±
Lung 16.6 + 3.29 9.86 + 0.57 7.45 + 2.06 5.56 ± 0.54
3.49 1.32
Spleen 4.63 + 0.56 4.76 ± 1.12 4.10 + 0.14 3.75 + 0.21 3.79 ± 0.89 3.23 ± 0.53
Kidneys 17.8 + 2.49 24.5 ± 4.38 28.8 + 1.49 24.7 ± 1.85 22.6 + 2.69 16.1 + 1.69
Stomach 3.19 + 0.95 2.86 ± 1.03 2.92 + 0.17 1.39 + 0.27 1.49 ± 0.48 0.91 + .010
Intestines 3.70 + 0.73 3.71 ± 1.09 3.70 + 0.40 2.19 ± 0.23 1.73 + 0.50 1.21 + 0.26
Liver 5.81 + 2.65 4.56 ± 1.18 4.87 + 0.42 3.35 ± 0.26 2.53 + 0.77 1.78 + 0.03
Salivary glands 5.60 + 0.70 5.02 ± 1.17 5.49 + 0.59 4.69 + 0.33 3.45 ± 1.09 2.19 ί 0.1
Muscle 1.91 + 0.16 2.04 ± 0.37 2.01 + 0.10 1.61 + 0.18 1.32 ± 0.41 0.91 ± 0.15
Bone 2.82 + 0.41 2.47 ±0.39 2.71 + 0.21 2.02 + 0.31 1.63 ± 0.56 1.07 ± 0.27
PC-3 PIP Tumor 8.49 + 0.62 19.9 ± 0.79 31.0 + 5.79 53.8 ± 5.61 72.3 + 24.7 75.7 + 2.46
PC-3 flu Tumor 3.84 + 1.10 5.32 ± 1.06 5.98 + 0.91 5.47 ± 0.67 5.69 + 3.65 3.52 + 0.54
Tumor-to-blood 0.31 ± 0.04 0.83 ± 0.10 1.32 ± 0.29 3.13 ± 0.35 5.94 + 1.53 10.3 + 0.57
Tumor-to-liver 1.64 + 0.61 4.60 ± 1.34 6.38 + 1.28 16.1 ± 1.75 29.0 + 5.26 42.6 + 1.90
Tumor-to-kidney 0.48 + 0.07 0.83 ± 0.14 1.07 + 0.18 2.17 + 0.06 3.17 + 0.84 4.7 ± 3.38
144 h.p.i. 192 h.p.i.
Blood 6.02 ± 0.60 5.29 ± 0.18
Heart 2.55 ± 0.25 2.08 + 0.14
Lung 4.99 ± 1.00 4.17 + 0.68
Spleen 2.94 + 0.39 3.13 + 0.90
Kidneys 1 1.2 ± 3.82 7.35 + 0.92
Stomach 0.73 ± 0.10 0.78 + 0.07
Intestines 0.88 + 0.18 0.96 ± 0.03
Liver 1.50 + 0.14 1.25 ± 0.20
Salivary glands 1.67 ± 0.27 1.55 + 0.06
Muscle 0.54 + 0.07 0.62 + 0.04 Bone 1.14 ± 0.23 0.83 + 0.22
PC-3 PIP Tumor 68.9 ± 8.80 58.9 + 12.4
PC-3 flu Tumor 2.77 + 0.41 2.42 ± 0.23
Tumor-to-blood 11.4 + 0.47 11.1 ± 1.97
Tumor-to-liver 46.0 ± 2.53 47.1 + 2.44
Tumor-to-kidney 6.44 + 1.27 7.97 ± 0.70
B i od i stri b uti on of 1 77 L u- PSMA-AL B -04 a n d 1 77 L u- PSMA-A L B -05
The tissue distribution of 177Lu-PSMA-ALB-04 and 177Lu-PSMA-ALB-05 was investigated over a period of eight days (Fig. 5B).
Blood activity levelsin animals injected with 177Lu-PSMA-ALB-04 was very high at early time points and remained by far the highest. A high PSMApos PC-3 PIP tumor accumulation was observed, which slightly decreased towards the end of the study. The accumulated activity in the PSMAneg PC-3 flu tumor and other non-target organs was clearly below blood levels, indicating highly PSMA-specific binding and uptake in vivo.
The high levels in the blood pool of animals injected with 177Lu-PSMA-ALB-05 were decreasing quickly and remained stable at low levels until the end of the study. Highest uptake of radioactivity could be observed in the PSMApos PC-3 PIP tumors of mice injected with 177Lu-PSMA-ALB-05, which was followed by a steady wash-out from the tumor tissue. The uptake in PC-3 flu tumors and other tissues was clearly below blood levels, indicating PSMA-specific binding and uptake in vivo. Biodistribution data for 177Lu-PSMA-ALB-04 and - 05 are shown in Table 4 and 5 below.
Table 4: Biodistribution of 177Lu-PSMA-ALB-04 in PC-3 PIP/flu Tumor-Bearing Mice
1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i.
46.7 ± 11.65 ±
Blood n/d n/d 55.3 ± 3.10 38.2 ± 1.48
9.64 1.22
Heart 16.4 ± 5.71 n/d n/d 22.1 ± 3.70 13.6 ± 0.36 4.81 ± 0.29
Lung 25.6 + 4.59 n/d n/d 40.1 ± 7.77 24.6 ± 2.02 9.96 ± 1.44
Spleen 11.0 ± 3.56 n/d n/d 13.6 ± 1.90 14.0 ± 1.70 6.76 ± 0.59
Kidneys 17.8 ± 4.49 n/d n/d 41.5 ± 1.44 38.2 ± 4.10 15.3 ± 1.49
Stomach 4.01 ± 0.62 n/d n/d 6.18 ± 0.95 5.04 ± 0.35 1.74 ± 0.11
Intestines 6.22 ± 1.1 1 n/d n/d 8.13 ± 1.31 7.27 ± 0.82 2.36 ± 0.17
Liver 29.3 ± 9.10 n/d n/d 17.6 + 2.04 13.1 ± 0.67 4.67 ± 0.82
Salivary glands 9.93 ± 2.33 n/d n/d 12.5 ± 0.42 10.6 ± 0.42 4.08 ± 0.44
Muscle 1.96 ± 0.40 n/d n/d 5.82 ± 1.62 4.62 ± 0.38 1.56 ± 0.56
Bone 4.74 ± 1.31 n/d n/d 8.88 ± 0.19 6.80 ± 0.67 2.54 ± 0.31
PC-3 PIP Tumor 9.56 ± 2.71 n/d n/d 82.8 ± 6.84 93.2 ± 12.4 61.4 + 7.68
PC-3 flu Tumor 3.46 ± 2.66 n/d n/d 14.0 ± 0.60 12.6 ± 0.82 5.63 ± 0.37
5.29 ±
Tumor-to-blood 0.20 ± 0.02 n/d n/d 1.50 ± 0.11 2.45 ± 0.41
0.74
Tumor-to-liver 0.33 ± 0.06 n/d n/d 4.76 ± 0.67 7.16 ± 1.16 13.4 ± 2.88
Tumor-to-kidney 0.54 ± 0.09 n/d n/d 2.00 ± 0.11 2.47 ± 0.50 4.03 ± 0.56
144 h.p.i. 192 h.p.i.
Blood n/d 3.75 ± 1.49
Heart n/d 1.73 ± 0.70
Lung n/d 4.10 ± 1.77
Spleen n/d 5.22 ± 3.17
Kidneys n/d 8.82 ± 3.83
Stomach n/d 0.56 ± 0.19
Intestines n/d 0.67 ± 0.15
Liver n/d 2.45 ± 0.81
Salivary glands n/d 1.96 ± 0.73
Muscle n/d 0.71 ± 0.54
Bone n/d 1.17 ± 0.61
PC-3 PIP Tumor n/d 57.6 ± 17.3
PC-3 flu Tumor n/d 3.31 ± 1.43
Tumor-to-blood n/d 15.8 ± 3.55
Tumor-to-liver n/d 23.9 ± 5.12
Tumor-to-kidney n/d 6.91 ± 2.24 Table 5: Biodistribution of 177Lu-PSMA-ALB-05 in PC-3 PIP/flu Tumor-Bearing Mice
1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 96 h p.i.
Blood 21.3 + 6.06 10.2 ± 1.98 n/d 1.67 ± 0.29 1.66 + 0.37 1.79 + 0.57
Heart 7.56 + 1.89 3.82 ± 0.63 n/d 0.65 + 0.11 0.54 + 0.15 0.70 ± 0.21
Lung 15.0 ± 1.24 7.07 + 1.44 n/d 1.80 ± 0.78 1.48 + 0.62 1.36 + 0.29
Spleen 5.78 + 1.40 3.29 + 0.74 n/d 1.13 + 0.23 0.71 ± 0.23 0.64 + 0.29
Kidneys 59.3 + 1.38 52.8 + 7.17 n/d 23.9 ± 4.02 12.8 + 2.62 6.89 + 0.31
Stomach 2.04 ± 0.43 1.15 ± 0.17 n/d 0.28 + 0.06 0.29 + 0.08 0.24 + 0.07
Intestines 2.71 ± 0.40 1.33 + 0.25 n/d 0.28 ± 0.05 0.28 ± 0.10 0.30 + 0.1 1
Liver 5.69 ± 1.59 2.96 + 0.50 n/d 0.82 ± 0.35 0.56 + 0.16 0.74 + 0.14
Salivary glands 6.17 + 2.12 2.75 + 0.72 n/d 0.49 + 0.10 0.45 + 0.10 0.46 + 0.10
Muscle 2.36 + 1.01 1.30 + 0.23 n/d 0.19 ± 0.06 0.20 ± 0.08 0.15 ± 0.06
Bone 3.03 ± 0.52 1.67 ± 0.27 n/d 0.31 ± 0.08 0.28 ± 0.05 0.28 ± 0.04
PC-3 PIP Tumor 46.9 ± 0.43 75.3 ± 15.3 n/d 79.4 + 11.1 60.3 + 10.7 45.0 + 7.94
PC-3 flu Tumor 3.72 + 0.83 2.10 + 0.20 n/d 0.59 ± 0.10 0.57 ± 0.09 0.49 ± 0.1 1
Tumor-to-blood 2.31 + 0.58 7.43 + 1.43 n/d 48.2 + 7.04 36.7 ± 1.81 27.1 + 10.0
Tumor-to-liver 8.65 + 2.21 25.6 + 4.58 n/d 106 + 28.6 110 + 12.2 62.8 ± 18.8
Tumor-to-kidney 0.79 + 0.02 1.42 + 0.19 n/d 3.38 ± 0.58 4.72 ± 0.18 6.51 + 0.98
144 h .p.i. 192 h .p.i.
Blood 1.75 ± 0.35 1.48 ± 0.13
Heart 0.65 ± 0.17 0.59 ± 0.05
Lung 1.25 ± 0.18 1.22 + 0.26
Spleen 0.56 ± 0.08 0.55 ± 0.10
Kidneys 4.28 + 0.26 2.70 + 0.36
Stomach 0.23 + 0.04 0.16 + 0.04
Intestines 0.27 + 0.05 0.24 + 0.04
Liver 0.72 ± 0.13 0.84 + 0.06
Salivary glands 0.46 ± 0.09 0.37 ± 0.04
Muscle 0.17 + 0.04 0.14 + 0.01
Bone 0.25 ± 0.05 0.26 + 0.04
PC-3 PIP Tumor 33.9 + 0.80 27.9 + 3.24
PC-3 flu Tumor 0.52 + 0.13 0.45 + 0.06
Tumor-to-blood 19.9 + 3.88 19.0 ± 2.97
Tumor-to-liver 47.9 + 8.28 33.4 + 1.64
Tumor-to-kidney 7.93 + 0.30 10.4 ± 0.25 Biodistribution of 177Lu-PSMA-ALB-06, 177L.u-PSMA-ALB-07, 177Lu- PSMA-ALB-08
The tissue distribution of 177Lu-PSMA-ALB-06, -07 and -08 was investigated up to three days post injection (Fig.5C).
Blood activity levels of all compounds decreased quickly and were comparable throughout the entire study. The highest PSMApos PC-3 PIP tumor accumulation was observed for compound 177Lu-PSMA-ALB-06, which slightly decreased towards the end of the study. The accumulated activity in the PSMAneg PC-3 flu tumor and other non-target organs was below blood levels, indicating PSMA-specific binding and uptake in vivoiov all compounds tested. Biodistribution data for 177Lu-PSMA-ALB-06, -07 and -08 are shown in Table 6, 7 and 8 below.
Table 6: Biodistribution of 177Lu-PSMA-ALB-06 in PC-3 PIP/flu Tumor-Bearing Mice
1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 48 h p.i. 72 h p.i.
Blood n/d 16.2 ± 1.40 n/d 1.49 + 0.50 n/d 0.62 + 0.06
Heart n/d 5.41 ±0.82 n/d 0.68 + 0.18 n/d 0.26 + 0.02
Lung n/d 9.40 ± 1.55 n/d 2.48 + 2.68 n/d 0.67 + 0.05
Spleen n/d 3.14 ±0.29 n/d 0.76 + 0.18 n/d 0.53 + 0.02
Kidneys n/d 18.9 ± 0.77 n/d 10.5 ± 2.13 n/d 5.58 + 0.62
Stomach n/d 1.89 ± 0.19 n/d 0.28 + 0.07 n/d 0.13 + 0.02
Intestines n/d 2.64 ±0.27 n/d 0.30 + 0.06 n/d 0.15 ± 0.00
Liver n/d 3.45 ±1.50 n/d 0.50 + 0.11 n/d 0.28 ± 0.02
Salivary glands n/d 3.26 ±0.16 n/d 0.52 + 0.11 n/d 0.24 + 0.03
Muscle n/d 1.60 ±0.38 n/d 0.21 + 0.04 n/d 0.07 + 0.02
Bone n/d 2.23 ±0.08 n/d 0.41 + 0.15 n/d 0.18 + 0.01
76.08 ±
PC-3 PIP Tumor n/d n/d 108 ± 11.6 n/d 77.9 ± 7.52
7.67
PC-3 flu Tumor n/d 3.16 ±0.39 n/d 0.79 + 0.23 n/d 0.43 + 0.03
Tumor-to-blood n/d 4.72 ± 0.51 n/d 77.6 ± 21.8 n/d 127 ± 24.9
24.29 ±
Tumor-to-liver n/d n/d 222 + 49.5 n/d 277 ± 19.3
8.27
Tumor-to-kidney n/d 4.02 ± 0.25 n/d 10.4 ± 1.16 n/d 14.1 ± 2.02
96 h.p.i. 192 h.p.i.
Blood n/d n/d
Heart n/d n/d
Lung n/d n/d
Spleen n/d n/d
Kidneys n/d n/d Stomach n/d n/d
Intestines n/d n/d
Liver n/d n/d
Salivary glands n/d n/d
Muscle n/d n/d
Bone n/d n/d
PC-3 PIP Tumor n/d n/d
PC-3 flu Tumor n/d n/d
Tumor-to-blood n/d n/d
Tumor-to-liver n/d n/d
Tumor-to-kidney n/d n/d
(n/d = not determined)
Table 7: Biodistribution of 177Lu-PSMA-ALB-07 in PC-3 PlP/flu Tumor-Bearing Mice
1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 72 h p.i. 96 h p.i.
Blood I n/d 6.67 ± 2.04 n/d 0.79 ± 0.08 0.40 ± 0.06 n/d
Heart j n/d 2.43 ± 0.78 n/d 0.40 ± 0.00 0.21 ± 0.01 n/d
Lung j n/d 4.67 ± 0.92 n/d 0.73 ± 0.06 0.43 ± 0.02 n/d
Spleen I n/d 3.41 ± 1.46 n/d 1.14 ± 0.04 0.49 ± 0.03 n/d
Kidneys j n/d 67.0 ± 9.50 n/d 51.9 ± 6.34 26.0 ± 1.58 n/d
Stomach j n/d 1.09 ± 0.30 n/d 0.18 ± 0.06 0.10 ± 0.01 n/d
Intestines j n/d 1.27 ± 0.45 n/d 0.20 ± 0.03 0.10 ± 0.01 n/d
Liver j n/d 1.94 ± 1.02 n/d 0.52 ± 0.04 0.44 ± 0.08 n/d
Salivary glands n/d 2.09 ± 0.50 n/d 0.43 ± 0.04 0.21 ± 0.01 n/d
Muscle i n/d 0.78 ± 0.22 n/d 0.13 ± 0.01 0.08 ± 0.01 n/d
Bone n/d 1.30 ± 0.27 n/d 0.31 ± 0.10 0.31 ± 0.06 n/d
63.5 ±
PC-3 PIP Tumor j n/d n/d 84.6 ± 14.2 62.6 ± 6.35 n/d
27.4
PC-3 flu Tumor | n/d 1.80 ± 0.27 n/d 0.80 ± 0.17 0.43 ± 0.04 n/d
9327 ±
Tumor-to-blood j n/d n/d 107 ± 12.2 160 ± 37.0 n/d
1.75
Tumor-to-liver j n/d 33.6 ± 6.56 n/d 162 ± 17.3 147 ± 38.4 n/d
Tumor-to-kidney j n/d 0.88 ± 0.28 n/d 1.64 ± 0.29 2.41 ± 0.18 n/d
144 h.p.i. 192 h.p.i.
Blood n/d n/d
Heart n/d n/d
Lung n/d n/d
Spleen n/d n/d
Kidneys n/d n/d
Stomach n/d n/d
Intestines n/d n/d
Liver n/d n/d
Salivary glands n/d n/d
Muscle n/d n/d
Bone n/d n/d PC-3 PIP Tumor n/d n/d
PC-3 flu Tumor n/d n/d
Tumor-to-blood n/d n/d
Tumor-to-liver n/d n/d
Tumor-to-kidney n/d n/d
(n/d = not determi ned)
Table 8: Biodistributi on of 177Lu-PSMA-ALB-08 in PC-3 PI P/flu Tumor-Bearing Mice
1 h.p.i. 4 h.p.i. 8 h p.i. 24 h p.i. 72 h p.i. 96 h p.i.
Blood n/d 0.41 ± 0.18 n/d 0.08 ± 0.01 0.06 + 0.02 n/d
Heart n/d 0.19 ± 0.07 n/d 0.04 ± 0.01 0.03 ± 0.01 n/d
Lung n/d 0.48 + 0.21 n/d 0.09 ± 0.02 1.28 ± 2.02 n/d
Spleen n/d 0.53 ±0.10 n/d 0.10 ± 0.03 0.11 ± 0.05 n/d
Kidneys n/d 27.2 ± 5.93 n/d 13.9 ± 2.32 7.98 ± 0.62 n/d
Stomach n/d 0.40 ± 0.25 n/d 0.03 ± 0.01 0.02 ± 0.01 n/d
Intestines n/d 0.20 ± 0.09 n/d 0.03 ± 0.01 0.02 ± 0.01 n/d
Liver n/d 0.27 ± 0.10 n/d 0.1 1 ± 0.02 0.12 ± 0.01 n/d
Salivary glands n/d 0.19 + 0.06 n/d 0.07 ± 0.05 0.03 ± 0.02 n/d
Muscle n/d 0.06 ± 0.03 n/d 0.02 ± 0.01 0.01 ± 0.01 n/d
Bone n/d 0.16 ± 0.03 n/d 0.07 ± 0.02 0.09 ± 0.05 n/d
PC-3 PIP Tumor n/d 46.9 ± 16.7 n/d 33.0 ± 5.04 24.1 ± 5.37 n/d
PC-3 flu Tumor n/d 0.25 ± 0.19 n/d 0.09 ± 0.05 0.09 ± 0.07 n/d
Tumor-to-blood n/d 116 ± 9.46 n/d 421 ± 45.7 416 ± 89.5 n/d
Tumor-to-liver n/d 177 ± 2.72 n/d 295 ± 40.1 207 ± 47.1 n/d
Tumor-to-kidney n/d 1.70 ± 0.22 n/d 2.39 ± 0.24 3.02 ± 0.68 n/d
144 h.p.i. 192 h.p.i.
Blood n/d n/d
Heart n/d n/d
Lung n/d n/d
Spleen n/d n/d
Kidneys n/d n/d
Stomach n/d n/d
Intestines n/d n/d
Liver n/d n/d
Salivary glands n/d n/d
Muscle n/d n/d
Bone n/d n/d
PC-3 PIP Tumor n/d n/d
PC-3 flu Tumor n/d n/d
Tumor-to-blood n/d n/d
Tumor-to-liver n/d n/d
Tumor-to-kidney n/d n/d
(n/d = not determ

Claims

Claims
1 . A compound according to General Formula (1 )(i) or (1 )(ii)
(D(i)
Figure imgf000118_0001
4
Linker Pbm
D— N— [CH2]a — CH
\
Spacer
Figure imgf000118_0002
(D(ii) wherein
Abm is an albumin binding entity,
Pbm is a PSMA binding entity,
D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7, 1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-(carboxyethyl)- benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4,7-triazacyclononane- 1 ,4,7-triacetic acid (NOT A), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazonan-1 - yl)pentanedioic acid (NODAGA), 2 -(4,7, 10-tris(carboxymethyl)-1 ,4,7, 1 0- tetraazacyclododecan-1 -yl)-pentanedioic acid (DOTAGA), 1 ,4,7-triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-1 -[methyl(2-carboxyethyl)- phosphinic acid]-4,7-bis[methyI(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9, 1 5-tetraazabicyclo[9,3, 1 ]pentadeca-1 (1 5), 1 1 , 1 3-triene-3,6,9-triacetic acid (PCTA), N'-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and
Diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,
X is each independently selected from O, N, S or P,
R1 and R2 are each independently selected from H, F, CI, Br, I, branched, unbranched or cycl ic G-C12 hydrocarbyl, C2-G2 alkenyl, C2-C12 alkylnyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, CONR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, or R1 and R2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic G -Go hydrocarbyl group, wherein said hydrocarbyl group is optional ly interrupted by up to 2 heteroatoms and optionally substituted by up to 3 groups independently selected from F, CI, Br, I, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, and SR7, =0, =S and =NH,
Y is selected from a single bond or a linear, branched or cyclic, optionally substituted G-G2 alkyl, optionally interrupted by up to two heteroatoms, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, wherein one or more of the non-adjacent CH2-groups may independently be replaced by -0-, -CO- -CO-O-, -O-CO-, -NR6-, -NR6-CO- -CO-NR6- -NR6-COO- - O-CO-NR6-, -NR6-CO-NR6-, -CH=CH- , -C≡C- -O-CO-O-, SR6-, SO3R6-,
R6 and R7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl,
R3, R4 and R5 are each independently selected from -COH, -C02H, -S02H, - SO3H, -S04H, -P02H, -PO3H, -PO4H2, -C(0)-(G-Go)alkyl, -C(O)-O(G-G0)alkyl, - C(0)-NHR8, or -C(0)-NR8R9' wherein R8 and R9 are each independently selected from H, bond, (G-Go)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH-benzyl, -C(0)-(G -Go)alkylene, -(CH2)P-NH, -(CH2)P-(G -Go)alkyene, -(CH2)P-NH-C(0)-(CH2)q, -(CHrCH2)t-NH-C(0)-(CH2)p, -(CH2)P-CO-COH, -(CH2)P-CO-C02H, -(CH2)P-C(0)NH- C[(CH2)q-COH]3, -C[(CH2)p-COH]3/ -(CH2)p-C(0)NH-C[(CH2)q-C02H]3/ -C[(CH2)P- C02H]3 or -(CH2)p-(C5-Ci4)heteroaryl,
the spacer comprises at least one C-N bond,
the linker is characterized by General Formula (6) as defined herein, and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10,
or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof.
2. The compound according to claim 1 , wherein said compound is characterized by General Formula (1 a):
Figure imgf000120_0001
wherein
D is a chelator, preferably selected from 1 ,4,7,1 0-tetraazacyclododecane- 1 ,4,7,1 0-tetraacetic acid (DOTA), N,N"-bis[2-hydroxy-5-
(carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1 ,4, 7- triazacyclononane-1 ,4, 7-triacetic acid (NOTA), 2-(4, 7-bis( carboxymethyl)-1 ,4, 7- triazonan-1 -yOpentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1 ,4,7,1 O-tetraazacyclododecan-1 -yOpentanedioic acid (DOTAGA), 1,4,7- triazacyclononane phosphinic acid (TRAP), 1 ,4,7-triazacydononane-1 -[methyl(2- carboxyethyOphosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1.]pentadeca-1(15),11, 13-triene-3,6,9- triacetic acid (PCTA), N'-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminope ntyO(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), and Diethylenetriaminepentaacetic acid (DTPA) or derivatives thereof,
X is each independently selected from O, N, S or P,
R1 and R2 are each independently selected from H, F, CI, Br, I, branched, unbranched or cyclic, optionally substituted, G-C12 hydrocarbyl, C2-C12 alkenyl, C2- C12 alkylnyl, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, CONR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, or R1 and R2 are joined to form a cyclic structure comprising a branched, unbranched or cyclic G-Go hydrocarbyl group, wherein said hydrocarbyl group is optionally interrupted by up to 2 heteroatoms and optionally substituted by up to 3 groups independently selected from F, CI, Br, I, OR6, OCOR6, COOR6, CHO, COR6, CH2OR6, NR6R7, CH2NR6R7, and SR7, =0, =S and =NH,
Y is selected from a single bond or a linear, branched or cyclic, optionally substituted G-G2 alkyl, optionally interrupted by up to two heteroatoms, OR6, OCOR6, CHO, COR6, CH2OR6 NR6R7, COOR6, CH2NR6R7, SR6, =0, =S or =NH, wherein one or more of the non-adjacent CH2-groups may independently be replaced by -0-, -CO- -CO-O-, -O-CO-, -NR6-, -NR6-CO- -CO-NR6-, -NR6-COO- - O-CO-NR6-, -NR6-CO-NR6-, -CH=CH- , -C≡C- -O-CO-O-, SR6-, SO3R6-,
R6 and R7 are each independently selected from H or branched, unbranched or cyclic G-12 hydrocarbyl,
R3, R4 and R5 are each independently selected from -COH, -CO2H, -SO2H, - SO3H, -SO4H, -PO2H, -PO3H, -PO4H2, -C(0)-(G-Go)alkyl, -C(O)-O(G-G0)alkyl, - C(0)-NHR8, or -C(0)-NR8R9' wherein R8 and R9 are each independently selected from H, bond, (C1-C10)alkylene, F, CI, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(0)-NH- benzyl, -C(O)-(G-G0)alkylene, -(CH2)P-NH, -(CH2)P-(G-G0)alkyene, -(CH2)P-NH-
C(0)-(CH2)q, -(CHrCH2)rNH-C(0)-(CH2)p, -(CH2)P-CO-COH, -(CH2)P-CO-C02H, - (CH2)p-C(0)NH-C[(CH2)q-COH]3, -C[(CH2)p-COH]3, -(CH2)p-C(0)NH-C[(CH2)q- C02H]3, -C[(CH2)p-C02H]3or -(CH2)p-(C5-Ci4)heteroaryl, the spacer comprises at least one C-N bond,
the linker is characterized by the Structural Formula (6):
Figure imgf000122_0001
(6) wherein
X is each independently selected from O, N, S or P,
Q is selected from substituted or unsubstituted alkyl, alkylaryl and cycloalkyl, preferably from substituted or unsubstituted C5-Ci4 aryl, C5-Ci4 alkylaryl or C5-C14 cycloalkyl, W is selected from -(CH2)c-aryl or -(CH2)c-heteroaryl, wherein c is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 , and a, b, p, q, r, t is each independently an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 1 0, or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof.
3. The compound according to claim 1 or 2, wherein the chelator D is selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof, most preferably, from DOTA, NODAGA, D03AP, D03APPrA or D03APABn.
4. The compound according to any one of claims 1 to 3, wherein each X is O.
5. The compound according to any one of claims 1 to 4, wherein Y is a linear or branched, optionally substituted, G-C12 hydrocarbyl, more preferably a linear or branched, optionally substituted, G-Go hydrocarbyl, even more preferably a linear or branched, optionally substituted, G-Qi hydrocarbyl, even more preferably a linear or branched, optional ly substituted, G-G hydrocarbyl. The compound according to claim 5, wherein Y is a linear C1 -C3 hydrocarbyl.
The compound according to any one of the preceding claims, wherein R1 and R2 are each independently selected from H and halogen, preferably iodine or bromine, and C1 -6 alkyl, preferably C1-3 alkyl, even more preferably methyl.
The compound according to claim 7, wherein in General Formula (1 ) the group
Figure imgf000123_0001
characterized by any one of Structural Formulas (2a), (2b) or (2c):
Figure imgf000123_0002
(2 a) (2 b)
Figure imgf000123_0003
(2 c)
The compound according to any one of the preceding claims, wherein R3, R4 and R5 are each independently selected from -COH, -C02H, -S02H, -SO3H, -SO4H, -PO2H, - PO3H, -PO4H2.
1 0. The compound according to claim 9, wherein each of R3, R4 and R5 are selected from -C02H.
1 1 . The compound according to any one of the preceding claims, wherein Q is selected from C5-C7 cycloalkyl. 12. The compound according to claim 1 1 , wherein Q is cyclohexyl.
1 3. The compound according to any one of the preceding claims, wherein W is selected from -(CH2)c-napthtyl, -(CH2)c-phenyl, -(CH2)c-biphenyl, -(CH2)c-indolyl, -(CH2)C- benzothiazolyl, wherein c is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0.
14. The compound according to claim 1 3, wherein W is -(CH2)-naphthyl. 1 5. The compound according to any one of the preceding claims, wherein the linker is characterized by Structural Formula (6a):
Figure imgf000124_0001
(6a) The compound according to claim 1 5, wherein said compound is characterized by General Formula (1 c):
Figure imgf000125_0001
(1 c) or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes thereof. 7. The compound according to any one of the preceding claims, wherein the spacer comprises a linear or branched, optionally substituted G -C20 hydrocarbyl, more preferably G -C12 hydrocarbyl, even more preferably C2-C6 hydrocarbyl, even more C2- C4 hydrocarbyl, the hydrocarbyl comprising at least one, optionally up to 4 heteroatoms preferably selected from N. 8. The compound according to claim 1 7, wherein the spacer comprises -[CHR10]u-NR - , wherein R10 and R11 are each be independently selected from H and branched, unbranched or cyclic C1 -G2 hydrocarbyl, and u is an integer selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0. 9. The compound according to claim 1 8, said compound being characterized by General Formula (7a):
Figure imgf000125_0002
R1 (7a) pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof.
The compound according to claim 1 9, said compound being characterized by Structural Formula (7a)(i) or (7a)(ii):
Figure imgf000126_0001
(7a)(i)
Figure imgf000126_0002
(7a)(ii) or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes of (7a)(i) or (7a)(ii).
21 . The compound according to any one of claims 1 to 1 6, wherein the spacer comprises at least one amino acid residue.
22. The compound according to claim 21 , said compound being characterized by General Formula (7b):
Figure imgf000127_0001
(7b) wherein
A is an amino acid residue,
V is selected from a single bond, N, or an optionally substituted G-C12 hydrocarbyl comprising up to 3 heteroatoms, wherein said heteroatom is preferably selected from N, n is an integer selected from 1 , 2, 3, 4 or 5
or pharmaceutical ly acceptable salts, esters, solvates or radiolabeled complexes thereof.
23. The compound according to claim 21 or 22, wherein said amino acid residue(s) is/are selected from (D-/L-) aspartate, glutamate or lysine.
24. The compound according to claim 23, wherein said spacer is characterized by Formula (3b) or Formula (3c):
Figure imgf000128_0001
(3 b) wherein m is an integer selected from 1 or 2, and n is an integer selected from 1 , 2, 3, 4 or 5, preferably from 1 , 2 or 3;
Figure imgf000128_0002
wherein o is an integer selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 1 0. 25. The compound accordi ng to any one of claims 21 to 24, said compound being characterized by Structural Formula (7b)(i), (7b)(ii) or (7b)(iii).
Figure imgf000129_0001
(7b)(i)
Figure imgf000129_0002
(7b)(iii)
Figure imgf000130_0001
or pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes of (7b)(i), (7b)(ii) or (7b)(iii).
26. Use of a compound according to any one of claims 1 to 25 for the preparation of a radiolabeled complex.
27. A radiolabeled complex comprising a radionuclide and a compound according to any one of the preceding claims.
28. The radiolabeled complex according to claim 27, wherein the metal is selected from the group consisting of 94Tc, 99mTc, 90ln, i ln, 67Ga, 68Ga, 86Y, 90Y, 177Lu, 151Tb, 186Re, 188Re, 64Cu, 67Cu, 55Co, 57Co, 43Sc, Sc, 47Sc, 225Ac, 213Bi, 212Bi, 212Pb, 227Th, 153Sm, 166Ho, 152Gd, 153Gd, 157Gd, or 166Dy.
29. A pharmaceutical composition comprising the compound according to any one of claims 1 to 25, or a radiolabeled complex according to any one of claim 27 or 28, and a pharmaceutically acceptable carrier and/or excipient.
30. A kit comprising a compound according to any one of claims 1 to 25 or a pharmaceutically acceptable salt, ester, solvate or radiolabeled complex thereof, a radiolabeled complex according to any one of claims 27 or 28 or a pharmaceutical composition according to claim 29.
31 . The compound according to any one of claims 1 to 25, the radiolabeled complex according to any one of claims 27 or 28, the pharmaceutical composition according to claim 29 or the kit according to claim 30 for use in medicine and/or diagnostics.
32. The compound according to any one of claims 1 to 25, the radiolabeled complex according to any one of claims 27 or 28, the pharmaceutical composition according to claim 29 or the kit according to claim 30 for use in a method of detecting the presence of cells and/or tissues expressing prostate-specific membrane antigen (PSMA).
33. The compound according to any one of claims 1 to 25, the radiolabeled complex according to any one of claims 27 or 28, the pharmaceutical composition according to claim 29 or the kit according to claim 30 for use in a method of diagnosing, treating and/or preventing prostate cancer, pancreatic cancer, renal cancer or bladder cancer.
34. The compound, radiolabeled complex, pharmaceutical composition or kit for the use according to any one of claims 31 to 33, wherein said use comprises
(a) administering said compound, radiolabeled complex or pharmaceutical composition to a patient, and
(b) obtaining a radiographic image from said patient.
35. An in vitro method of detecting the presence of cells and/or tissues expressing prostate- specific membrane antigen (PSMA) comprising
(a) contacting said PSMA-expressing cells and/or tissues with a compound, radiolabeled complex, pharmaceutical composition or kit according to any one of the preceding claims;
(b) applying detection means, optionally radiographic imaging, to detect of said cells and/or tissues.
36. The compound, radiolabeled complex, pharmaceutical composition or kit for the use according to claim 31 to 34, or the method according to claim 34, wherein radiographic imaging comprises positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
37. The compound, radiolabeled complex or pharmaceutical composition for the use according to claim 31 to 34 or 36, or the method according to claim 35, wherein said one or more cells or tissues comprise (optionally cancerous) prostate cells or tissues, (optionally cancerous) spleen cells or tissues, or (optionally cancerous) kidney cells or tissues.
38. The compound, radiolabeled complex or pharmaceutical composition for the use according to claim 31 to 34 or 36, or the method according to any one of claims 35 to 37, wherein the presence of PSMA-expressing cells or tissues is indicative of a prostate tumor (cell), a metastasized prostate tumor (cell), a renal tumor (cell), a pancreatic tumor (cell), a bladder tumor (cell), and combinations thereof.
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