WO2013060793A1

WO2013060793A1 - Bifunctional ligands for radiometals

Info

Publication number: WO2013060793A1
Application number: PCT/EP2012/071180
Authority: WO
Inventors: Hans-Jürgen Peter WESTER; Johannes NOTNI; Jakub SIMECEK
Original assignee: Technische Universität München
Priority date: 2011-10-25
Filing date: 2012-10-25
Publication date: 2013-05-02

Abstract

The present invention relates to the field of nuclear medicine and molecular imaging as well as targeted therapy and targeted radiotherapy, i.e. radiopharmaceuticals for imaging and targeted radiotherapy using metal ion radionuclides in combination with chelators that are functionalized with targeting vectors or additional signalling units.

Description

Bifunctional ligands for radiometals Technical field

The present invention relates to the field of nuclear medicine and molecular imaging, i.e. radiopharmaceuticals for imaging and targeted radiotherapy using metal ion radionuclides or paramagnetic metal ions in combination with bifunctional chelators.

Background art

Metal radionuclides are currently used in nuclear imaging and therapy. Related radiopharmaceuticals (often called tracers) are usually bioconjugates, that is, they are formed from a metal binding group (chelate ligand), which is covalently bound, with or without additional bridging molecular units (so-called linkers), to one or more molecular units displaying biological activity, e.g. affinity to certain tissues (so-called targeting vectors).

Currently, DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid) is the most used chelate ligand for this purpose. It forms stable complexes with many transition metal ions as well as lanthanide ions. Very frequently, one of the acetic acid side arms of this molecule is transferred into a secondary or tertiary amide, bearing the linker or the targeting vector. For imaging purposes, the metal ion is added as the last step, thereby forming the complex which serves as the tracer.

Particularly for complexation of lanthanide ions, the most established chelators are DOTA and derivatives of this structure, such as 1,4,7, 10-tetraazacyclododecane- 1,4,7-triacetic acid (DO3A).

The coordination of the metal ion occurs on the nitrogen atoms of the azamacrocycle backbone and on the deprotonated carboxyl groups of the acetic acid substituents. These carboxylic acid moieties thus have to be deprotonated in order to act as coordination sites. Therefore, a pH value exceeding their pKg of approx. 3.5-4.5 must be maintained during the labelling procedure. Labelling at a lower pH is substantially hampered. In addition, labelling of DOTA-like structures requires either heating, usually up to 80-95 °C, or comparably high ligand concentrations (in the range of 1 mM).

In order to prepare bioconjugates of chelators, the use of protecting groups on either side is mandatory in most cases. Particularly in case of DOTA or DO3A, the

carboxylate moieties intended for metal complexation have to be protected during amide coupling. The tris-tert-butyl esters of these compounds are thus employed for conjugation, requiring an additional subsequent deprotection step in order to obtain the desired conjugate. Recently, it has been shown that chelating systems based on polyazacycloalkanes (particularly 1,4,7-triazacyclononane) bearing methylenephosphinic acid substituents on the nitrogen atoms possess particularly favorable complexation properties towards metal ions, such as Ga³⁺, in comparison to carboxylate functionalized chelators like DOTA. Radiolabeling, that is, complexation of the radionuclide ⁶⁸Ga³⁺, was done in a wide range of pH values (pH 0.5 to 5), using very low concentrations of the chelate ligand. Furthermore, a compound of this class, possessing methyl-(2- carboxyethylphosphinic acid) N substituents, has been functionalized with a variety of biomolecules, such as peptide bioligands, and used for PET imaging (Notni, 1;

Hermann, P.; Havlickova, J.; Kotek, J,; Kubicek, V.; Plutnar, J.; Loktionova, N.; Riss, P. J.; Rosch, F.; Lukes, I., Chem. Eur. J. 2010, 16, 7174-7185).

Aims of the invention

The invention aims at chelate ligands that can form complexes with non-radioactive as well as radioactive metal ions. A further aim of the invention is the possibility of simple preparation of conjugates of said chelate ligands with other functional molecules such as linkers and/or biomolecules. Furthermore, the invention aims at the preparation of ligands that posses one or more addressing units, e.g. peptides, proteins, small molecules or other units, that bind with high affinity to a given molecular target being overexpressed in a pathological state or disease. The invention furthermore aims at coordination compounds (metal ion chelates) of said ligands with radioactive metal ions that can be applied in nuclear imaging techniques such as gamma scintigraphy, single photon emission computed tomography (SPECT), or positron emission tomography (PET), as well as in targeted radiotherapy.

Disclosure of the invention

The invention relates to chelators based on 1,4,7-triazacycloalkanes, bearing one substituted methylenephosphinic acid moiety at one nitrogen atom, and other substituents on the other nitrogen atoms. The present invention therefore relates to compounds (chelators) according to general formula (I),

wherein m is selected from integers 1, 2, 3, 4, 5, or 6; n, o, p are independently of each other selected from integers 1 and/or 2; X, Y are independently of each other selected from the group consisting of hydrogen, as well as linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals, but excluding the radical -CH₂-P(O)(OH)-(CH₂)₂- COOH. Preferably m, n, o, and p have the following meanings:

m is 1, n is 1, o is 1, and p is 1, or

m is 2, n is 1, o is 1, and p is 1, or

m is 3, n is 1, o is 1, and p is 1.

In another preferred embodiment of compounds according to general formula (I), the variables n, o, p are equalling 1, and the variable m is equalling 2. Therefore, the present invention preferably relates to compounds according to general formula (I), wherein n is 1, o is 1, p is 1, and m is 2, and X and Y have the same meanings as defined in general formula (I).

In another preferred embodiment of compounds according to general formula (I), X and Y are hydrogen, or methyl, or ethyl, or propyl, or isopropyl, or phenyl, or hydroxy methyl, or benzyl or tert-butoxycarbonyl (BOC) radicals. Therefore, the present invention preferably relates to compounds according to general formula (I), wherein X and Y are independently of each other selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, hydroxymethyl, benzyl, tert- butoxycarbonyl (BOC) radicals, and the variables m, n, o, and p have the same meanings as defined in general formula (I).

Compounds according to general formula (I) are generally suitable for chelating metal ions. However, substituents X and Y can also contain additional atoms and molecular entities suitable for coordination to metal ions. These additional coordination sites generally improve the stability of metal chelates. Compounds according to general formula (I) are therefore of particular importance as precursors for the synthesis of other compounds to be used as chelate ligands, to which the present invention further relates.

Therefore, in another preferred embodiment of compounds (chelators) according to general formula (I), the substituents X and Y are carboxylic acid radicals, such as carboxymethyl, 2-carboxyethyl, 3-carboxypropyl radicals, or corresponding carboxylic acid ester radicals. The invention therefore further relates to compounds (chelators) according to general formula (I), wherein X and Y are independently of each other selected from the group consisting of carboxylic acids or carboxylic acid ester radicals, according to the general formula (Ila),

wherein m is selected from integers 1, 2, 3, 4, 5, or 6; n, o, p are independently of each other selected from integers 1 and 2; r, s are independently of each other selected from integers 1, 2, and 3; R¹, R² are independently of each other selected from the group consisting of hydrogen, as well as linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals.

In a preferred embodiment of compounds (chelators) according to general formula (Ila), the variable m is equalling 2, the variables n, o, p, r, and s are equalling 1. The invention therefore preferably relates to compounds (chelators) according to general formula (Ila), with the variable m equalling 2, the variables n, o, p, r, and s equalling 1, and R¹ and R² having the same meanings as defined in general formula (Ila). In another preferred embodiment of compounds (chelators) according to general formula (Ila), the substituents R¹ and R² are hydrogen, methyl, ethyl, benzyl, tert- butyl, or triphenylmethyl. The invention therefore preferably relates to compounds (chelators) according to general formula (Ila), with R¹ and R² independently of each other being selected from the group consisting of hydrogen, methyl, ethyl, benzyl, tert-butyl, or tri phenyl methyl radicals, and the variables m, n, o, p, r, and s having the same meanings as defined in general formula (Ila).

In a particularly preferred embodiment of compounds (chelators) according to general formula (Ila), the variable m is equalling 2, the variables n, o, p, r, and s are equalling 1, and the substituents R¹ and R² are tert-butyl radicals. The invention therefore particularly preferably relates to compounds (chelators) according to general formula (Ila), with the variable m equalling 2, the variables n, o, p, r, and s equalling 1, and R¹ and R² being tert-butyl radicals.

In another preferred embodiment of compounds (chelators) according to general formula (I), the substituents X and Y are substituted methyl(phosphinic acid) radicals. The invention therefore further relates to compounds (chelators) according to general formula (I), wherein X and Y are independently of each other selected from the group of substituted methyl(phosphinic acid) radicals, according to general formula (Ilia),

wherein m is selected from integers 1, 2, 3, 4, 5, or 6; n, o, p are independently of each other selected from integers 1 and/or 2; R³, R⁴ are independently of each other selected from the group consisting of hydrogen, as well as linear or cyclic,

substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals, but excluding the radical -(CH₂)2-COOH. In a preferred embodiment of compounds (chelators) according to general formula (Ilia), the variable m is equalling 2, and the variables n, o, and p are equalling 1. Therefore, the present invention preferably relates to compounds (chelators) according to general formula (Ilia), wherein m is 2, n is 1, o is 1, and p is 1, and R³ and R⁴ have the same meanings as defined in general formula (Ilia). In another preferred embodiment of compounds (chelators) according to general formula (Ilia), R³ and R⁴ are hydrogen, or methyl, (hydroxy) methyl, isopropyl or phenyl radicals. Therefore, the present invention preferably relates to compounds (chelators) according to general formula (Ilia), wherein R³ and R⁴ are independently of each other selected from the group of hydrogen or methyl, (hydroxy)methyl, isopropyl and phenyl radicals, and the variables m, n, o, and p have the same meanings as defined in general formula (Ilia).

Particularly preferred is the compound (chelator) according to general formula (Ilia), with the variable m equalling 2 and the variables n, o, and p equalling 1, wherein R³ and R⁴ are hydroxymethyl radicals. Therefore, the present invention particularly preferably relates to a compound (chelator) according to general formula (Ilia) as mentioned above, wherein m is 2, n is 1, o is 1, and p is 1, and R³ and R⁴ are hydroxymethyl radicals (-CH₂OH).

The terminal carboxylic acid group of compounds according to general formula (I), and therefore also according to general formulae (Ila) and (Ilia), can be reacted with primary or secondary amines to form the corresponding amides. Useful reagents for this procedure are uronium-like coupling reagents, such as HBTU (0- Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate), TBTU (0- (Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), and HATU (2- (lH-7-Azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uranium hexafluorophosphate Methanaminium), or COMU ((l-Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate). The phosphinic acid moieties are in general not affected by this coupling protocol. Particularly, it was found that no formation of any phosphinamide is observed. It must be noted that, according to the invention, the use of the coupling reagents TBTU and HBTU on methyl(2-carboxyethylphosphinic acid) N substituents is identical to a method described in prior art {Chem. Eur. J. 2010, 16, 7174-7185).

Therefore, the present invention further relates to compounds (chelators) according to general formulae (II) and (III),

wherein m is selected from integers 1, 2, 3, 4, 5, or 6; n, o, p are independently of each other selected from integers 1 and/or 2; r, s are independently of each other selected from integers 1, 2, and 3; R¹, R² are independently of each other selected from the group consisting of hydrogen, as well as linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals; R³, R⁴ are independently of each other selected from the group consisting of hydrogen, as well as linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals, but excluding the radical -(CH₂)2-COOH; Z is either OH or an amide residue -NR'R", wherein R' and R" are independently of each other selected from hydrogen or the group consisting of signalling units such as fluorophors, metal chelators, or residues with high affinity to a molecule or molecular structure pathologically or normally expressed in tissues, e.g. antigens, receptors, transporters, enzymes, or organic molecules, e.g. amino acids, peptides, proteins, carbohydrates, nucleobases, antibodies, antibody fragments, and mixtures thereof.

In a preferred embodiment of compounds according to general formulae (II) and (III), R' and R" are independently of another biomolecules selected from the group consisting of c(RGDfK)(Pbf,tBu), c(DGRKf)(Pbf,tBu), cyclo(d-Tyrl-d-Orn2-Arg3-Nal4- Gly5) linked via D-Orn2 (CPCR4), H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys- Thr-ol (Disulfide bridge: 2-7), linked via D-Phel, H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr- OH (Disulfide bridge: 2-7), linked via D-Phel, H-D-Phe-Cys-BzThi3-D-Trp-Lys-Thr- Cys-Thr-OH (Disulfide bridge: 2-7, linked via D-Phel, H-D-Phe-Cys-BzThi3-D-Trp-Lys- Thr-Cys- Thr-ol (Disulfide bridge: 2-7), linked via D-Phel, H-D-Phe-Cys-1-Nal3-D-Trp- Lys-Thr-Cys-Thr-OH (Disulfide bridge: 2-7), linked via D-Phel, H-D-Phe-Cys-1-Nal3- D- Trp-Lys-Thr-Cys-Thr-ol (Disulfide bridge: 2-7), [Lys40(Ahx-DTPA)NH2]-exendin-4, [Lys40(Ahx-DOTA)NH2]-exendin-4, [Lys4,Phe7,Pro34]NPY (linked via Lys4)

Demobesin 1, panbombesin, minigastrin 11 and 9, Demogastrin, CCK8 (nonsulfated), pan-somatostatin (KE-88), 4-N02-Phe-(D)Cys-Thr-Trp-Lys(Dde)-Thr-Cys-(D)Tyr-OH (Disulfide bridge: 2-7), AcTZ14011 and T140, folic acid, pteroic acid, nitroimidazole derivatives, and mixtures thereof.

In another preferred embodiment of compounds (chelators) according to general formulae (II) and (III), R' and/or R" are small, bifunctional molecules which contain at least one additional functional group allowing for further functionalisation (so- called linkers). Such functional groups can be carboxylic acid, carboxylic acid ester, amine, carbamoyl ester, terminal alkyne, terminal alkene, azide, cyanide, thiol, isothiocyanate, aldehyde, succinimide, maleimide, oxy-amines, hydrazide, terminal bromide, terminal iodide. The invention therefore preferably relates to compounds (chelators) according to general formulae (II) and (III), wherein R' and/or R" belong to this group of substituents.

In a particularly preferred embodiment of compounds according to general formulae (II) and (III), R' is hydrogen and R" is selected from the above mentioned residues. The present invention further relates to a process for the preparation of compounds according to general formulae (II) and (III) by reaction of compounds of general formula (Ila) and (Ilia), respectively, with compounds H-NR'R", wherein R' and R" have the same meanings as defined above.

This process according to the present invention can in general be conducted under conditions that are known to the skilled artisan. In a preferred embodiment this process according to the present invention is conducted in the presence of at least one coupling agent, for example selected from uronium-like coupling reagents, such as HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate), TBTU (0-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HATU (2-(lH-7-Azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uronium hexafluorophosphate Methanaminium), or COMU ((l-Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate). The present invention further relates to coordination compounds (chelates) according to general formulae (IV) and (V),

(k-3)

wherein m is selected from integers 1, 2, 3, 4, 5, or 6; n, o, p are independently of each other selected from integers 1 and/or 2; r, s are independently of each other selected from integers 1, 2, and 3; R³, R⁴ are independently of each other selected from the group consisting of hydrogen, as well as linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals, but excluding the radical -(CH₂)2-COOH; Z is either OH or an amide residue -NR'R", wherein R' and R" are independently of each other selected from hydrogen or the group consisting of signalling units such as fluorophors, metal chelators, or residues with high affinity to a molecule or molecular structure pathologically or normally expressed in tissues, e.g. antigens, receptors, transporters, enzymes, or organic molecules, e.g. amino acids, peptides, proteins, carbohydrates, nucleobases, antibodies, antibody fragments, and mixtures thereof; M^k+ is a metal ion or radiometal ion, wherein A- denotes its oxidation state.

In compounds (chelates) according to general formulae (IV) and (V), M^k+ is usually coordinated by the oxygen atoms of the carboxylate or the phosphinate groups, and preferably also by the tertiary nitrogen atoms of the macrocycle.

In a preferred embodiment of compounds (chelates) according to general formulae (IV) and (V), ^k+ is selected from the group consisting of Sc³⁺, Y³⁺, Ga³⁺, In³⁺, Pd²⁺, Ti⁴⁺, Zr⁴⁺, Al³⁺, Cr³⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, Co³⁺, Ni²⁺, Fe ⁺, Fe³⁺, Ca²⁺, Mg²⁺, Be²⁺, Cd²⁺, Ag⁺, Nb³⁺, Lu³⁺ and mixtures thereof.

The present invention therefore preferably relates to compounds (chelates) according to general formulae (IV) and (V), wherein M^k+ is selected from the group consisting of Sc³⁺, Y³⁺, Ga³⁺, In³⁺, Pd²⁺, Ti⁴⁺, Zr⁴⁺, Al³⁺, Cr³⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, Co³⁺, Ni²⁺, Fe²⁺, Fe³⁺, Ca²⁺, Mg²⁺, Be²⁺, Cd²⁺, Ag⁺, Nb³⁺, Lu³⁺ and mixtures thereof.

In a further preferred embodiment of compounds (chelates) according to general formulae (IV) and (V), M is selected from radioisotopes, for example selected from the group consisting of ⁴Sc, ⁶Sc, ⁴⁷Sc, ⁵⁵Co, ^99mTc, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁱⁿIn, ^113mIn, ^114mIn, ⁹⁷Ru, ⁶²Zn, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶³Cu, ⁶⁴Cu, ⁶⁷Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ⁹⁰Y, ¹⁰⁹Pd, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁰⁵Rh, ^lnAg, ⁸⁸Zr, ⁸⁹Zr, and mixtures thereof. In this case, k \s selected from integers 2, 3, and 4. Particularly preferably M is selected from the group consisting of ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, and mixtures thereof.

The present invention therefore preferably relates to compounds (chelates) according to general formulae (IV) and (V), wherein M is selected from the group consisting of ⁴⁴Sc, ⁴⁶Sc, ⁴⁷Sc, ⁵⁵Co, ^99mTc, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ^mIn, ^113mIn, ^114mIn, ⁹⁷Ru, ⁶²Zn, ⁶⁰Cu ⁶¹Cu, ⁶²Cu, ⁶³Cu, ⁶⁴Cu, ⁶⁷Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ⁹⁰Y, ¹⁰⁹Pd, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁰⁵Rh, ⁱⁿAg, ⁸⁸Zr, ⁸⁹Zr, and mixtures thereof, and k \s selected from integers 2, 3 and 4, and particularly preferably relates to compounds (chelates) according to general formulae (IV) and (V), wherein M is selected from the group consisting of Ga, Ga, Cu, and mixtures thereof.

Particularly preferred are metal chelates of compounds according to general formulae

(IV) and (V), wherein M^k+ is either ⁶⁸Ga³⁺ or ⁶⁴Cu²⁺. The present invention therefore particularly relates to these chelates.

According to this preferred embodiment, compounds of general formulae (IV) and

(V) are labelled with radioisotopes, where in this context, the term labelling is referring to binding of the radioactive metal ions by the chelator by means of complex formation.

The present invention therefore further relates to a process for the preparation of compounds according to general formulae (IV) and (V) by reacting compounds of general formulae (Ila), (Ilia), (II), and (III), with compounds comprising the metal cation M^k+.

Compounds comprising the metal cation ^k+ are preferably solutions of metal salts comprising M^k+ and various anions, selected from the group of sulfates, fluorides, clorides, bromides, nitrates, phosphates, carbonates, hydrogencarbonates, sulfonates, acetates, acetylacetonates, and mixtures thereof.

This process according to the present invention is in general conducted under usual conditions for reactions of this kind which are known to the skilled artisan. In a preferred setting, the process is conducted at temperatures ranging from ambient temperature (room temperature) to 37 °C.

A further central aim of the invention is the use of the described metal complexes for molecular imaging and radiotherapy. The present invention therefore further relates to the method of using compounds (chelates) according to general formulae (IV) and (V) in molecular or nuclear imaging as well as in radiotherapy.

A preferred method of using compounds (chelates) according to general formulae (IV) and (V) in molecular and nuclear imaging is the use in magnetic resonance imaging (M I), gamma scintigraphy, single photon emission computed tomography (SPECT) or positron emission tomography (PET) and combinations thereof.

The present invention therefore preferably relates to the method of using compounds according to general formulae (IV) and (V), wherein the molecular or nuclear imaging is magnetic resonance imaging (MRI), gamma scintigraphy, single photon emission computed tomography (SPECT) or positron emission tomography (PET) and combinations thereof.

By using metal complexes of metal radionuclides, particularly those emitting also alpha or beta radiation (for example, ⁶⁴Cu), application in targeted radiotherapy is possible. The invention therefore relates to the method of using compounds according to general formulae (IV) and (V), in targeted radiotherapy, or in targeted radiotherapy in combination with magnetic resonance imaging (MRI) and/or gamma scintigraphy and/or single photon emission computed tomography (SPECT) and/or positron emission tomography (PET).

Experimental section and the examples

General

Analytical HPLC was performed using a Sykam HPLC system with low-pressure gradient mixer, equipped with a Nucleosil C18-RP column (100 x 4.6 mm, 5 pm particle size), at a flow rate of 1 ml/min. Eluents were water and acetonitrile, both containing 0.1% trifluoroacetic acid (TFA). Two gradients were used: Gradient A, 20- 80% MeCN in 24 min and Gradient B, 30-60% MeCN in 12 min.

If not mentioned else, preparative HPLC was done using a Sykam system with two separate solvent pumps, equipped with a YMC C18ec column (250 x 30 mm, 5 prn particle size), at a flow rate of 20 ml/min. Solvents were similar to analytical HPLC. Separations were generally done with eluent compositions individually optimized for each compound (see below). ESI- S was measured on a Varian LC-MS system.

Radio-HPLC was performed on a Sykam system using a Chromolith column (Merck, 100x4.6 mm) with radioactivity and UV detection (220 nm). Eluents were water (A) and acetonitrile (B), both containing 0.1% TFA (isocratic elution with 3% B for 2 min, followed by a gradient to 60% B in 6 min and isocratic elution with 95% B for 3 min).

Peptide synthesis

The protected cyclic peptides (for examples, see scheme below) were prepared according to literature protocols, using to the skilled artisan known standard methods for solid phase peptide synthesis (Fmoc strategy).

- 13 -

General synthetic procedure for coupling of amines using HATU

Molar amounts of reagents used are given below for each synthesis and individual compounds. NOPO, diisopropylethylamine (DIPEA) and the amine (peptide) were dissolved in DMSO. Then HATU was added with stirring. RP-HPLC was used for reaction control. After the reaction had finished (usually within 10 minutes), the reaction mixtures were diluted with water and subjected to diafiltration with 0.05 M NaCI solution and then pure water (Amicon stirred cell, membrane with 0.5 kDa MWCO), followed by lyophilisation of the cell contents, and finally purified by preparative HPLC. Fractions containing the product were collected, partly evaporated, freeze-dried and characterized by HPLC and MS.

If one of the reactants contained the protecting group, deprotection step was performed with freeze-dried and re-dissolved intermediate, deprotected conjugate was the purified by preparative HPLC.

General procedure for removal of acid-sensitive protecting groups (Pbf, tBu)

The dry compound was dissolved in 0.5-1 ml trifluoroacetic acid and allowed to react for 1 h. Then, the mixture was slowly added to diethyl ether, the precipitate separated by centrifugation and dried in vacuo. If necessary, purification was done using preparative HPLC, followed by concentration of the eluates and lyophilization to yield the final products.

General procedure for removal of protecting group Dde

The dry compound was dissolved in 0.5-1 ml 0.2% hydrazine hydrate and allowed to react for 15 minutes. Then, the mixture was slowly added to diethyl ether, the precipitate separated by centrifugation and dried in vacuo. If necessary, purification was done using preparative HPLC, followed by concentration of the eluates and lyophilization to yield the final products.

Synthesis of chelating moieties

As an example of synthesis of compounds of general formula (Ilia), two synthetic routes to l,4,7-triazacyclononane-l,4-N,N ^'-bis(methylen(hydroxomethylphosphinic acid))-7-N-methylene(2-carboxyethyl)phosphinic acid) (NOPO) are shown.

Synthetic path A:

l-benzyl-l,4,7-triazacyclononane (1.28 g, 5.88 mmol) and paraformaldehyde (0.44 g, 14.67 mmol) were mixed at LT in 50% aq. H₃PO₂ (6.6 ml, 60.61 mmol) and water (5 ml). Reaction mixture was stirred for 12 hours at LT, then purified on cationic exchanger DOWEX 50 in proton cycie. Triazacyclononane-based products were eluted with HC EtOH 1:1. Eluate was evaporated and crude product was dissolved in HCI, paraformaldehyde (0.73 g, 24.33 mmol) was added and solution was refluxed for 5 hours, then evaporated and purified on silicagel, fractions containing product were collected, evaporated to give 1.20 g of dark-yellow solid which was then dissolved in water, 10% Pd/C (0.4 g) was added and mixture was stirred under H₂ atmosphere. After 12 hours, mixture was filtered on frit S4, evaporated and controlled by H and ³¹P NMR spectroscopy. Crude product A (0.9 g) and 2-(carboxyethyl)phosphinic acid (0.53 g, 3.8 mmol) were dissolved in HCI and heated. Paraformadehyde (0.35 g, 11.73 mmol) was added. Reaction mixture was co-evaporated with water and then poured on DOWEX 50 in H⁺ form. Product was eluted with water, evaporated and left under vacuum in desiccator over P₂O₅ to give NOPO-0.6 H₂O in form of yellowish oil which solidifies upon standing. Overall yield 0.53 g, 18 %. Yield of the last step 40 %.

Synthetic path B: mviso

TMSO -COOTMS

(B)

2-(carboxyethyl)phosphinic acid (0.13 g, 0.9 mmol) was dissolved in

hexamethyldisilazane (5 ml), the glassware was filled with argon, and the solution was heated to 150°C for 24 hours. (A) (0.10 g, 0.3 mmol) was dissolved by heating with HMDS at 130°C under argon atmosphere and the reaction mixture containing (B) was added as one portion in the syringe. Paraformaldehyde (0.030 g, 1.0 mmol) was added in one portion and the reaction mixture was stirred for 24 hours, then cooled to the laboratory temperature. Content of the reaction flask was dissolved in water and loaded onto small column filled with DOWEX 50 in H⁺ form. Product was eluted with water in neutral fraction, which was then evaporated and left under vacuum in desiccator over P₂0₅ to give NOPO-0.6 H₂O in form of yellowish oil which solidifies upon standing. Yield 70 mg, 47 %.

³¹P NMR {^XH} (121 MHz, D₂O): d 34.72 (s, 2P), d 40.31 (s, IP)

3¹P NMR (121 MHz, D₂O): d 34.69 (s, 2P), d 40.28 (s, IP)

H NMR (600 MHz, D₂O): d 3.58 (s, ring CH₂, 4H), d 3.61 (s, ring CH₂, 2H), d 3.495 (d, 7/"=6.36 Hz, -N-CH.2-P-, 2H), d 3.83 (d, J/ =5.79 Hz, -P-CH₂-OH, 4H), d 3.50 (d, _7/ =5.55 Hz, -N-CH.2-P-, 4H), d 2.09 (m, -P-CH₂- CH₂-, 2H), d 2.69 (m, -P-CH₂- CH₂-, 2H)

¹³C NMR (150 MHz, D₂O): δ 52.29 (s, ring CH₂, 2C), δ 52.39 (s, ring CH₂, 4C), δ 53.82 (d, J ^c=86.56 Hz, N-CH₂-P, 2C), δ 60.20 (d, J ^C= 112.88 Hz, -P-CH₂-OH, 2C), δ 55.38 (d, J ^c=90.41 Hz, N-CH₂-P, 1C), δ 25.51 (d, J ^c=94.70 Hz, -P-CH₂-CH₂-, 1C), δ 27.52 (d, J ^c=3.52 Hz, -P-CH₂-CH₂-, 1C), δ 177.91 (d, J₃ ^PC= 12.55 Hz, -C=O, 1C) MS (ESI, positive,

496 [NOPO + H⁺]

Elemental analysis: found - C, 32.31; H, 6.67; N, 8.22; calculated - C, 33.22; H, 6.61; N, 8.30; best fits for C14H33.2N3O10.eP3 (NOPO^■ 0.6 H₂O)

Another example, where reaction pathway B can be used, is the synthesis of 1,4,7- triazacyclononane-l,4-N,N ^'-bis(tert-butylacetic acid)-7-N-methylen(2- carboxyethyl)phosphinic acid) (tBu₂-NOP2A)

2-(tert-butyloxycarbonyl)ethyl]phosphinic acid (0.260 g, 1.9 mmol) was dissolved in hexamethyldisilazane (5 ml), the glassware was filled with argon, and the solution was heated to 150°C for 24 hours. t-Bu₂NO₂A (0.200 g, 0.56 mmol) was dissolved separately in HMDS (7 ml) and added into solution with (B) via syringe,

paraformaldehyde (0.050 g, 1.6 mmol) was added in one portion, the reaction mixture was heated at 130 °C for 24 hours and then cooled to 25 °C. Each portion of yellow oil obtained (200 mg) was dissolved in 1 ml of water, filtered through a syringe filter (0.5 pm) and purified using semi-preparative HPLC system composed of LCD 50K gradient pump with a spectrophotometric detector LCD 2083 (ECOM), detection at wavelength 210 nm. Separations were performed on a semi-preparative column Luna RP8, 10 pm, 250x4.6 mm (Phenomenex) equipped with a Security Guard system (Phenomenex) holding a C8 cartridge. The mobile phases were mixed separately and degassed in a sonicator. Solution A: 20 % MeCN, 20 % 0.1M NH₄OAc and 60 % H₂O; solution B: 33 % MeCN, 20 % 0.1M NH₄OAc and 47 % H₂O; solution C: 55 % eCN, 20 % 0.1M NH₄OAc and 25 % H₂0 (solution C). Flow rate 20 ml/min. Gradient: start 100 % of A, after 19 minutes 100 % of B, after 1 minute 100 % of C, 10 minutes 100 % of C, 1 minute of A and 14 minutes of A. The fraction containing product was collected, evaporated in vacuo and freeze-dried to give tBu₂-NOP2A. Yield 0.130 g, 46 %.

³¹P NMR {¹H} (121 MHz, D₂0): d 32.42 (s, 1 P)

3¹P NMR (121 MHz, D₂O): d 32.55 (s, 1 P)

¹H NMR (600 MHz, D₂O): d 2.89 (bs, ring CH₂, 4H), d 3.12 (bs, ring CH_.2, 4H), d 3.35 (bs, ring <¾, 4H), d 3.63 (s, -N-CH₂-CO-, 4H), d 1 .49 (s, -CH₃, 18H), d 3.30 (d, J₂ ^PH=7A6 Hz, N-CH₂-P-, 2H), d 1.87 (m, -P-CH₂- CH₂-, 2H), d 2.41 (m, -P-CH₂- CH₂-, 2H)

¹³C {¹H} NMR (150 MHz, D₂O): δ 47.56 (s, ring CH₂, 2C), δ 49.66 (s, ring CH₂, 2C), δ 53.30 (s, ring CH₂, 2C), δ 56.62 (s, -N-CH₂-CO-, 2C), δ 172.98 (s, -N-CH₂-CO-, 2C), δ 84.27 (s, -C-(CH₃)₃, 2C), δ 28.03 (s, -CH₃, 6C), δ 53.77 (d, ^^ΡΟ=88.00 Hz, -N-CH₂- P-, 1 C), δ 27.63 (d,

Hz, -P- CH₂- CH₂-, 1 C), δ 181 .29 (d, J₃ ^PC=16.7 Hz, -COOH, 1 C)

MS (ESI, positive, [m/z]): 508 [NOP-2A + H⁺]

Analytical HPLC: Figure 1. Isocratic 52 % H₂0, 20 % 0,1M NH₄OAc, 28 % MeCN.

Ga:NOPO complex was prepared by dissolving the equimolar amounts of NOPO and gallium chloride in water. For characterisation, the complex was purified on weak cationic exchanger. Figure 2: ⁷¹Ga NMR spectra of Ga-NOPO. Figure 3: MS spectra of Ga-NOPO.

Synthesis of the conjugates and their Ga(III) complexes

NOPO-RGD

NOPO-0.6 H₂0 (24.8 mg, 49 μηιοΙ) and Arg(Pbf)-Asp(tBu)-cRGDfK FA (50 mg, 44 prnol) were dissolved in DMSO (0.5 ml), DIPEA (86 μΙ, 63.8 mg, 494 pmol) was added. HATU (57.2 mg, 150 pmol) was added and the solution was stirred for 10 minutes, meanwhile it turned to dark yellow. Precipitate resulting from addition of reaction mixture to NaCI solution was centrifuged, precipitate was dissolved in water and ultrafiltrated. Supernatant was evaporated, freeze-dried, deprotected by stirring 10 minutes with TFA (80 %, 1 ml), precipitated by addition into diethylether, centrifuged, dissolved in water and purified by preparative HPLC. Fractions containing the product were checked by MS, evaporated and freeze-dried to give 21 mg of NOPO-RGD, yield 40 %.

Preparative HPLC: Gradient 27-37 % MeCN in 30 min. Rt(NOPO-RGD) = 13.5-14.5 min Characterisation figure 4: Gradient A. Rt(NOPO-RGD) = 6.5 min.

MS (ESI, positive, [m/z]): 1081 [NOPO-RGD + H⁺]

MS (ESI, negative, [m/z]): 1079 [NOPO-RGD - H⁺]

Gallium complexation: 2mM solution of Ga(NO₃)3 (0.1 ml) and 2 mM solution of NOPO-NOC (0.1 ml) were mixed and heated at 95 °C for 5 minutes.

MS (ESI, positive, [m/z]): 1047 [Ga: NOPO-RGD + H⁺]

NOPO-NOC

Dde-NOC FA (10.0 mg, 7.4 pmol) was added to NOPO (5.5 mg, 11.1 pmol) in DMSO (total 0.5 ml), DIPEA (14.3 mg, 19.3 μΙ, 0.111 mmol) and HATU (21.1 mg, 55.5 pmol) were added, stirred 10 minutes at r. t, then analyzed by HPLC and transferred into sodium chloride solution. No precipitation occured so the solution was transferred into the ultrafiltration cell and ultrafiltrated. The supernatant was freeze-dried. Crude product was dissolved in DMF (1 ml) and hydrazine hydrate (31.2 ,ul, 2% in total volume) was added, the solution was stirred for 15 minutes, then freezed in liquid nitrogen and freeze dried.

Crude product was dissolved in water (0.3 ml) and TFA (0.3 ml) was added, solution was stirred 10 minutes, then other 0.5 ml of TFA was added and stirred for 30 minutes. The solution was transferred into diethylether and evaporated with addition of water, then freeze dried. Crude product was dissolved in water and purified using preparative HPLC with the gradient MeCN (0.1 % TFA) 25-45 % in 60 minutes. The product was eluted between 31 and 32 minutes. Crude product was dissolved in water (0.5 ml) and purified by preparative HPLC: Gradient 25-45 % MeCN in 60 min. The fraction containig product were collected, evaporated and freeze-dried. Yield 5.9 g (51 %).

Characterisation: figure 5. Gradient B. Rt(NOPO-NOC) = 8.5 min.

MS (ESI, positive, [m/z]): 1528 [M-H2O+H⁺], 774 [M-H2O +2H⁺]

Gallium complexation: 2 mM solution of Ga(NO₃)3 (0.1 ml) and 2 mM solution of

NOPO-NOC (0.1 ml) were mixed and heated at 95 °C for 15 minutes. MS (ESI, positive, [m/z]): 1612 [L+Ga3+-2H⁺], 806 [L+Ga3+-H⁺]

NOPO-Amb-CPCR4 CPCR4-Amb (41.0 mg, 0.034 mmol), NOPO (37.2 mg, 0.075 mmol) and DIPEA (0.13 ml, 96.75 mg, 0.75 mmol) were dissolved in DMSO (0.5 ml), HATU (85.73 mg, 0.225 mmol) was added and the solution was stirred for 10 minutes. Solution was HPLC analysed after 10 minutes and for control after 30 minutes as well, which did not show any difference from 10 minutes. Water was added and the solution was transferred into ultrafiltration cell and let filtered overnight. HPLC of eluate and supernatant were performed. Supernatant was freeze-dried. Crude product was treated in TFA (80%, 1 ml) for 30 minutes, precipitated by addition of diethylether, centrifuged twice with ether, dried and purified by preparative HPLC

chromatography. Yield 22.7 mg (51%).

MS (ESI, positive, [m/z]): 1299 [M+H⁺], 650 [M+2H⁺]

MS (ESI, negative, [m/z]): 1297 [M-H⁺]

Preparative HPLC: Isocratic 27 % MeCN. Rt(NOPO-AmB-CPCR4) = 15-16 min.

Characterisation: Gradient A. Rt(NOPO-Amb-CPCR4)=10.5 min.

Gallium complexation: 2 mM solution of Ga( 03)3 (0.1 ml) and 2 mM solution of NOPO-Amb-CPCR4 (0.1 ml) were mixed and heated at 95 °C for 5 minutes. MS (ESI, positive, [m/z]): 1365 [M+Ga3+-2H⁺], 683 [M+Ga3+-H⁺]

NOPO-TATE and NOPO-TOC

The conjugates were prepared in the same manner as NOPO-NOC. ⁶⁸Ga labelling

Eluate obtained from ⁶⁸Ge/⁶⁸Ga generator with Sn0₂ matrix (obtained from IDB Holland bv) was used without further purification. pH was adjusted by addition of HEPES to reach pH 3.3. 90μΙ of ⁶⁸Ga solution was mixed with 10 μΙ of chelator stock solution. Such solution was heated at 94 °C for 5 minutes, then cooled down in water bath, and evaluated by TLC (1M NH₄OAc:MeOH 1:1 on Varian chromatography paper, 0.1M sodium citrate on silica gel). The results of labelling experiments are depicted in figure 6.

⁶⁸Ga automated labelling

⁶⁸Ga-labelling was performed on an automated system (GallElut+ module from Scintomics, Fiirstenfeldbruck, Germany), carrying out the following steps. ⁵⁸Ga was obtained from a generator with Sn0₂ matrix (manufactured by IThembaLABS, South Africa, distributed by IDB Holland) which was eluted with 1.0 M HCI.

The precursor (e.g. NOPO-RGD, 0.5 nmol) was placed in a 4 ml conical reaction vial (AIITech), together with a solution of 260 mg 2-(4-(2-Hydroxyethyl)-l-piperazinyl)- ethansulfonsaure (HEPES) in 220 μΙ Water. Then a 1.25 ml fraction of the generator eluate, containing the hightest activity (approx. 900 MBq) was added, resulting in pH 1.8. The vial was heated to 100 °C for 5 min. Then the reaction mixture was passed over a SPE cartridge (Waters SepPak C8 light), the cartridge purged with 10 ml of water to remove free ⁶⁸Ga³⁺, inorganic ions and HEPES, and purged with air. The product was eluted with 2 ml of a 1:1 mixture of ethanol and water and the cartridge and lines purged with 1 ml of water. For animal experiments, 1 ml of PBS (pH 7.4) was added and the solution concentrated in vacuo to 1 ml, thus removing all ethanol and producing a formulation suitable for injection. Radiochemical yields (RCY) and specific activities of the ⁶⁸Ga-labelled NOPO-conjugates are listed in the table below.

Table: Radiochemical yields and specific activity (GBq/pmol) of labelled TRAP conjugates.

NOPO-RGD NOPO-Amb-CPCR4

n (nmol)

RCY ± SD SA ± SD RCY ± SD SA ± SD

1 96.4 ± 0.2 705.3 ± 29.2 95.7 ± 0.2 773.3 ± 52.2

0.5 96.3 ± 0.9 1507 ± 93.1 94.8 ± 0.3 1534.3 ± 101.2

0.1 77.2 ± 2.1 5555 ±

482.3 73.4 ± 10.6 5612.3 ± 510.6

Maximal achieved SA was ~6200 GBq/pmol.

Evaluation of the tracers in vitro/in vivo

logP determination - general procedure

Octanol-water partition coefficients were determined by addition of ca. 50 kBq of the respective labelled compound to Eppendorf cup containing each 500 μΙ of 1-octanol and isotonic phosphate buffered saline (PBS). After 2 min of vigorous stirring, the phases were separated by centrifugation, 100 pi aliquots of each phase taken out and the activity contained determined with a gamma counter. Each experiment was repeated 5 - 8 times.

NOPO-RGD

In vitro binding assay

Binding assays were done for NOPO-RGD, natGa-NOPO-RGD and also for echistatin, ¹²⁵I-FC131 and ¹⁹F-galacto-RGD to act as standards.

Determination of integrin receptor affinity was carried out using M21 human melanoma cells, possessing high ανβ3 expression. Experiments were carried out in 24-well plates. Ca. 2xl0⁵ cells were seeded into wells containing RPMI 1640 media and incubated for 24 h at 37 °C and 5% CO₂. Then the medium was exchanged with 0.5 ml, binding buffer (20 mmol/l Tris, pH 7.4, 150 mmol/l NaCI, 2 mmol/l

CaCI₂*2H₂0, 1 mmol/l MgCI₂*6H₂0, 1 mmol/l MnCI₂*4H₂0, 0.1% (m/m) BSA), containing 30.000-50.000 cpm ¹²⁵I-echistatin and NOPO-RGD in increasing concentrations from 10 ⁿ-10^"4 M. After incubation at room temperature for 2 h, the supernatant is removed, the cells washed twice with PBS, lysed with 1 M NaOH (1 ml) and the lysates counted for 60 s in a gamma counter. Experiments were performed at minimum three times in duplicates and IC₅₀ values calculated using GraphPad prism for sigmoidal (dose-response) regression analysis.

In the table below IC₅₀ values and logP values for the standard, NOPO-RGD and Ga(III)-NOPO-RGD are given (see Synthesis section and figure 7).

Compound ICso (riM) logP

Echistatin 0.98 ND

¹²⁵I-FC131 4.1 ND

¹⁹F-Galacto-RGD 319 -3.2

⁶⁸Ga-NOPO-RGD 995 -4.64

ND - not determined

Biodistribution of Ga-NOPO-RGD

Biodistribution studies were performed using CD-I athymic nude mice bearing human melanoma xenografts on both shoulders (right: M21 cell line with high ανβ3 integrin expression, left: M21L cell line with low ανβ3 integrin expression). The mice were injected approximately 15-20 MBq of ⁶⁸Ga-NOPO-RGD (specific activity: ca.

1700 GBq/pmol). After the specified time points (60 and 120 min, respectively), the mice were sacrificed, the organs taken out and counted in a gamma counter. For blockade, the mice were administered 100 pg (approx. 5 mg/kg) of unlabeled NOPO- RGD 10 min before tracer injection.

Figure 8 shows uptake values (given as percent injected dose per gram tissue) 90 min after tracer injection, as well as 120 min after injection with blockade.

In vivo stability for ⁶⁸Ga-NOPO-RGD

Metabolite studies were performed using mice similar as used for biodistribution. The mice were anaesthesized with isoflurane and injected approximately 40 MBq of ^Ga- NOPO-RGD. After 30 min the animals were sacrificed, the blood was collected in a syringe and centrifuged. The respective organs were removed, frozen with liquid nitrogen and homogenized by means of a ball mill. The resulting powder was suspended in 0.5-1 ml_ of PBS, stirred for 1 min, and centrifuged. For both organs and blood, the supernatant (plasma, respectively) was separated, both the pellet and supernatant counted in a gamma-counter in order to determine extraction efficiency or blood cell binding. Supernatants, plasma and urine were subjected to ultrafiltration (30 kDa MWCO) and analyzed by radio-TLC. Drop of ⁶⁸Ga-NOPO-RGD as a standard, liver, kidney, tumour, blood and urine sample were blundered on silica-coated Varian chromatography paper and let evoke with 1M ammonium acetate, then dried and pressed onto erased BAS-IP MS 2025 Imaging Plate (Fujifilm, Japan) which was then irradiated overnight in X-ray cassette, and then developed using CR35BIO reader and Aida image analyser v 4.24.

The table below shows extraction efficiencies for the tissues worked up with the above described procedure; the fraction of the blood activity in the plasma after centrifugation is given.

Mouse 1 extract pellet efficiency

CPM %

blood 1123477 319363 78%

liver 360046 23570 94%

kidney 945679 200716 82%

tumour M21 511977 69716 88%

Figure 9: Radio-TLC (1M NH₄OAc:MeOH 1:1, Varian chromatography paper): 1 - ⁶⁸Ga-NOPO-RGD as a standard, 2 - liver, 3 - kidney, 4 - tumour, 5 - blood, 6 - urine.

Mouse 2 extract pellet efficiency

without tumour CPM %

blood 1288275 225476 85%

liver 1306722 317841 80%

kidney 972779 99223 91%

Figure 10: Radio-TLC (Mouse 2): 1 - Ga-NOPO-RGD as a standard, 2 - blood, 3 - urine, 4 - kidney, 5 - liver.

Ga-NOPO-RGD tracer after synthesis and isolated for metabolic stability study. Figure 11: TLC on Varian paper: 0.1M NH₄OAc:MeOH (1:1).

Figure 12: TLC on Varian paper: 1M trisodium citrate.

Figure 13: Radio-HPLC.

Figure 14: HPLC (the same as QC) - metabolites in blood.

Figure 15: HPLC: Metabolites - kidneys.

Figure 16: HPLC: Metabolites - liver.

Figure 17: HPLC: Metabolites - urine.

PET Imaging

Preclinical imaging was done with mice similar as used for biodistribution. The mice were anaesthesized with isoflurane, placed in the PET camera, and 13-14 MBq of ⁶⁸Ga-NOPO-RGD (without blockade and with prior injection of 100 Mg of unlabelled NOPO-RGD) were administered via tail vein injection. The calculated molar amount of labelled tracer per injection was ca. 20 pmol with a calculated minimum specific activity of ca. 900 GBq/μιηοΙ. PET scans were recorded dynamically for 90 min.

Figure 18 shows PET images derived from data measured 75 min after injection (recording time: 15 min; maximum intensity projections; left: ⁶⁸Ga-NOPO-RGD, right: NOPO-RGD with blockade).

NOPO-NOC

LogP was determined as desribed above giving the value of -1.82.

Biodistribution of ⁶⁸Ga-NOPO-NOC

68Ga-NOPO-NOC for animal experiments were formulated in the same manner as described for automated labelling of NOPO-RGD.

Figure 19: Radio-HPLC of prepared ⁶⁸Ga-NOPO-NOC.

Biodistribution studies were performed using CD-I athymic nude mice bearing AR42J tumours. The mice were injected approximately 15-20 MBq of ⁶⁸Ga-NOPO-NOC (specific activity: ca. 500 GBq/pmol). After 90 minutes, the mice were sacrificed, the organs taken out and counted in a gamma counter. For blockade, the mice were administered 100 Mg of unlabeled NOPO-NOC 10 min before tracer injection.

Figure 20 shows uptake values (given as percent injected dose per gram tissue) 90 min after tracer injection, as well as 120 min after injection with blockade.

In vivo stability for ⁶⁸Ga-NOPO-NOC

In vivo stability of ⁶⁸Ga-NOPO-NOC as described for ⁶⁸Ga-NOPO-RGD. Evaluation was done using paper TLC with mobile phase ammonium acetate:MeOH 1:1. Healthy mouse extract pellet effciency

(nr.4) CPM %

blood 1205656 109437.6 92%

liver 409507.3 196765.7 68%

kidney 1405047 524515.5 73%

Figure 21: TLC evaluation of metabolic stability of ^Ga-NOPO-NOC in vivo. PET Imaging

Preclinical imaging of mice bearing AR42J tumours was done in the same way as described for ⁶⁸Ga-NOPO-RGD. The mice were anaesthesized with isoflurane, placed in the PET camera, and approximately 10 MBq of ⁶⁸Ga-NOPO-NOC (without blockade and with prior injection of 100 g of unlabelled NOPO-NOC) were administered via tail vein injection. The calculated specific activity of ca. 500 GBq/pmol. PET scans were recorded dynamically for 90 min. Figure 22: PET imaging with ⁶⁸Ga-NOPO-NOC (dynamic scan).

NOPO-CPCR4

LogP was determined as described above giving the value of -2.82. Competition binding studies were performed using Jurkat cells. ¹²⁵I-FC131 was used as a standard ligand. Samples containing 4-105 cells were incubated 2 hours at RT with gentle agilation (200mot/min), in PBS/0.2% BSA with ¹²⁵I-FC131 (100 000 cpm ~ 0.1 nM) in presence of increasing concentration (10-11 to 10-5 M) of FC131 as reference compound and natGa-NOPO-Amb-CPCR4, total volume 0.25 ml. After 2 hours, the tubes were centrifuged (5 min, 447 g, Megafuge 1.0, Heraeus Thermo Scientific) and the supernatant was removed, samples were washed twice with 0.4 ml of cold PBS. The amount of bound ¹²⁵I-FC131 was quantified with γ-counter. IC₅₀ value was determined using PRISM 4 software (Graph Pad Software, San Diego, CA). The determined IC₅o value was 730 nM.

Cu(II) complexes and ⁶⁴Cu labelling

100 μΙ of ⁶⁴Cu in 0.1M HCI was diluted with water (9.9 ml). 90 μΙ of that solution was added to 10 μΙ of ligand solution resulting in ligand concentrations 0.1, 0.3, 1, 3 and 10 μΜ at pH 3. Chelators were labelled at 25°C for 5 minutes and then analysed with TLC (0.1 M EDTA/silicagel); figure 23.

Labelling of NOPO-RGD at pH 5.7, 25 and 37°C in 1M NaOAc as well as HEPES lead in all the conditions to labelling >99% after 10 minutes. As a proof of stability of Cu-NOPO-like compounds, stability of ^MCu-NOPO-RGD was examined by addition of 1M EDTA to the solution of fully labelled conjugate. No transmetallation/decomposition was observed after 120 minutes.

Characterisation of Cu-NOPO and Cu-NOPO-conjugates

The copper(II) complexes were prepared by mixing the equimolar amount of Cu(N03)2 solution with solutions of NOPO and its conjugates. Products were identified by MS. Figures 24 to 27: Characterisation MS spectra (from the top): Cu- NOPO ([M ] 555), CU-NOPO-CPCR4 ([M ] 1357), Cu-NOPO-NOC ([M⁺] 1607, [M²⁺] 805), Cu-NOPO-RGD ([M ] 1140).

Claims

1. A compound according to general formula (I)

wherein m is selected from integers 1, 2, 3, 4, 5, or 6; and n, o and p are independently of each other selected from integers 1 and/or 2; and X and Y are independently of each other selected from the group consisting of hydrogen and linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals, but excluding the radical -CH₂-P(0)(OH)-(CH₂)2-COOH.

A compound according to the general formula (II)

wherein m is selected from integers 1, 2, 3, 4, 5, and 6; and n, o, and p are independently of each other selected from integers 1 and 2; and r and s are independently of each other selected from integers 1, 2, and 3; and R¹ and R² are independently of each other selected from the group consisting of hydrogen and linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals; and Z is either OH or an amide residue -NR'R", wherein R' and R" are independently of another selected from hydrogen or the group consisting of signalling units such as fluorophors, metal chelators, or residues with high affinity to a molecule or molecular structure pathologically or normally expressed in tissues, e.g. antigens, receptors, transporters, enzymes, or organic molecules, e.g. amino acids, peptides, proteins, carbohydrates, nucleobases, antibodies, antibody fragments, and mixtures thereof.

A compound according to the general formula (III)

wherein m is selected from integers 1,

2,

3,

4,

5, and 6; and n, o, and p are independently of each other selected from integers 1 and 2; and R³ and R⁴ are independently of each other selected from the group consisting of hydrogen or linear or cyclic, substituted or unsubstituted, aliphatic, heteroaliphatic, aromatic, heteroaromatic, saturated or unsaturated radicals, but excluding the radical -(CH₂)2-COOH; and Z is either OH or an amide residue -NR'R", wherein R' and R" are independently of each other selected from hydrogen or the group consisting of signalling units such as fluorophors, metal chelators, or residues with high affinity to a molecule or molecular structure pathologically or normally expressed in tissues, e.g. antigens, receptors, transporters, enzymes, or organic molecules, e.g. amino acids, peptides, proteins, carbohydrates, nucleobases, antibodies, antibody fragments, and mixtures thereof.

The compounds according to any of claims 2 and 3, wherein R' and R" are independently of each other selected from hydrogen or the group consisting of c(RGDfK)(Pbf,tBu), c(DGRKf)(Pbf,tBu), cyclo(d-Tyrl-d-Orn2-Arg3-Nal4-Gly5) linked via D-Orn2 (CPCR4), H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys- Thr-ol (Disulfide bridge: 2-7), linked via D-Phel, H-D-Phe-Cys-Phe-D-Trp-Lys-Thr- Cys-Thr-OH (Disulfide bridge: 2-7), linked via D-Phel, H-D-Phe-Cys-BzThi3-D- Trp-Lys-Thr-Cys-Thr-OH (Disulfide bridge: 2-7, linked via D-Phel, H-D-Phe- Cys-BzThi3-D-Trp-Lys-Thr-Cys- Thr-ol (Disulfide bridge: 2-7), linked via D- Phel, H-D-Phe-Cys-l-Nal3-D-Trp-Lys-Thr-Cys-Thr-OH (Disulfide bridge: 2-7), linked via D-Phel, H-D-Phe-Cys-1-Nal3-D- Trp-Lys-Thr-Cys-Thr-ol (Disulfide bridge: 2-7), [Lys40(Ahx-DTPA)NH2]-exendin-4, [Lys40(Ahx-DOTA)NH2]- exendin-4, [Lys4,Phe7,Pro34]NPY (linked via Lys4) Demobesin 1,

panbombesin, minigastrin 11 and 9, Demogastrin, CCK8 (nonsulfated), pan- somatostatin (KE-88), 4-N0₂-Phe-(D)Cys-Thr-Trp-Lys(Dde)-Thr-Cys-(D)Tyr-OH (Disulfide bridge: 2-7), AcTZHOll and T140, folic acid, pteroic acid, nitroimidazole derivatives, and mixtures thereof.

The compound according to any of claims 1 to 4, having one of the following structural formulae:

- 30 -

- 31 -

10 PCT/EP2012/071180

6. A process for the preparation of compounds according to any of claims 2 to 5 by reaction of compounds according to claim 1 with compounds H-NR'R", wherein R' and R" have the meanings as defined in any of claims 2 and 3.

7. A coordination compound comprising at least one compound according to any of claims 1 to 5 and at least one metal ion or radiometal.

8. The coordination compound according to claim 7, wherein at least one metal ion is selected from the group consisting of Sc³⁺, Y³⁺, Ga³⁺, In³⁺, Pd²⁺, Ti⁴⁺, Zr⁴⁺ Al³⁺, Cr³⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, Co³⁺, Ni²⁺, Fe²⁺, Fe³⁺, Ca²⁺, Mg²⁺, Be²⁺, Cd²⁺, Ag⁺, Nb³⁺ and mixtures thereof, the metal ion being preferably selected from the group consisting of Ga³⁺, Cu ⁺ and Al³⁺.

9. The coordination compound according to claim 7, wherein at least ones

radioisotope is selected from the group consisting of ⁴⁴Sc, ⁴⁶Sc, ⁴⁷Sc, ⁵⁵Co, 9^9mTc, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁱⁿIn, ^113mIn, ^114mIn, ⁹⁷Ru, ⁶²Zn, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶³Cu, 6⁴Cu, ⁶⁷Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ⁹⁰Y, ¹⁰⁹Pd, ¹⁷⁷Lu, ¹⁰⁵Rh, ⁱA_g/ ⁸⁸Zr, ⁸⁹Zr, and mixtures thereof, the radiometal being preferably selected from the group consisting of ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu.

10. The coordination compound according to any of claims 7 to 9, having one of the following structural formulae, wherein M represents any radiometal chosen the group of ⁶⁷Ga³⁺, ⁶⁸Ga³⁺, ⁶⁴Cu²⁺:

- 35 -

-36-

-37-

-38-

11. A process for the preparation of compounds according to any of claims 7 to 10 by reacting at least one compound according to any of claims 1 to 5 with compounds comprising at least one metal cation M^k+, wherein k denotes the oxidation state of the metal.

12. The process according to claim 10, which is conducted at low temperatures, preferably at temperatures ranging between ambient temperature (room temperature) and 37 °C, particularly preferably at ambient temperature (room temperature).

13. A compound according to any of claims 1 to 5 and 7 to 10 for use in a method of molecular or nuclear imaging.

14. A compound according to any of claims 1 to 5 and 7 to 10 for use in a method of molecular or nuclear imaging according to claim 13, wherein the molecular or nuclear imaging is magnet resonance imaging (MRI), gamma scintigraphy, single photon emission computed tomography (SPECT), positron emission tomography {PET), fluorescence imaging, Cherenkov imaging, or any combination thereof.

15. A compound according to any of claims 1 to 5 and 7 to 10 for use in a method of targeted therapy and targeted radiotherapy.

16. A compound according to any of claims 1 to 5 and 7 to 10 for use in a method of targeted therapy and targeted radiotherapy according to claim 15 in combination with any method for molecular or nuclear imaging according to any of claims 13 or 14.