IN VIVO STABLE HG-197(M) COMPOUNDS, METHOD FOR THE PRODUCTION THEREOF AND USE THEREOF IN NUCLEAR MEDICAL DIAGNOSTICS AND ENDORADIONUCLIDE THERAPY (THERANOSTICS)

The present invention relates to in vivo stable 197(m)Hg compounds according to formula (E) for use in nuclear medical diagnostics and endoradionuclide therapy (theranostics), particularly the treatment of cancer, a method for the production of the 197(m)Hg compounds comprising the step of radiolabeling of organic precursor compounds with NCA 197(m)Hg by electrophilic substitution; and the use of the 197(m)Hg compounds for nuclear medical diagnostics and endoradionuclide therapy (theranostics), particularly the treatment of cancer.

STATE OF THE ART

There has been a continuing need for effective radioisotopes in nuclear medical diagnostics and endoradionuclide therapy (theranostics).

The interest in the mercury isotope197(m)Hg was awakened primarily by the decay characteristics of both nuclear isomers, like convenient half-life197(m)Hg (T1/2=23.8 h, Eγ134 keV, 34%) and197Hg (T1/2=64.14 h, Eγ77 keV, 19%), low energy gamma radiations useful for diagnosis and numerous Auger and conversion electrons with high potential for cancer therapy.

Mercury (Hg) radioisotopes with low specific activity have been used for imaging from the 1950s (Greif et al., 1956, Sodee 1964) until the late 1960s (Matricali, 1969) exemplary for brain scanning and cancer imaging. Greif et al. disclose the use of197Hg labelled Neohydrin® as radionuclide in nuclear medical diagnostics of the kidney (Greif et al. 1956). The197Hg labelled Neohydrin® was produced by n/gamma reaction of enriched196Hg in a reactor, wherein a low specific activity of 1 GB/μmol was achieved. Furthermore, the product was contaminated with203Hg.

Alternatively, Walther et al. proved the feasibility of the production of the no carrier added (NCA) radionuclide197mHg from gold at low proton energies in sufficient quantity and quality for imaging and experimental therapeutic purposes (Walther et al. 2015). The production of the no carrier added (NCA) radionuclide197mHg was carried out through proton induced nuclear reactions on gold via the197Au(p,n)197(m)Hg reaction in quantities up to about each 100 MBq, wherein Au superseded the expensive enrichment for the target material. For separation of197(m)Hg and197Hg from the predominant part of the target material a liquid-liquid extraction method was applied. Walther et al. discloses a resin based method for the separation of Hg radionuclides from Au targets via di-(2-ethylhexyl)orthophosphoric acid (HDEHP) on an inert support (Walther et al. 2016). Advantageously, the separation method exhibits a higher separation factor, a better handling and the possibility for automation, which significantly improves radiation protection, significantly lower product losses during the separation, and convenient recycling of the gold target material.

The use of radionuclides in nuclear medical diagnostics and endoradionuclide therapy (theranostics) requires the production of in vivo stable labeling units. For the clinical chelation therapy of mercury poisoning the sulfur-containing chelating agents meso-dimercaptosuccinic acid (DMSA, Chemet®) and dimercaptopropanesulfonic acid (DMPS, Dimaval®) are generally used (George et al. 2004). However, George et al. discloses the instability of the formed Hg chelate complexes with DMSA and DMPS.

Thus, there remains a need for in vivo stable197(m)Hg compounds.

Griffith et al. discloses the organometallic mercury compound Chlormerodrin ((3-Carbaoylamino-2-methoxypropyl)-chloromercury, Neohydrin®), a mercurial diuretic, which was used in the treatment of chronic congestive heart failure (Griffith et al. 1956). Its radiolabeled derivative203Hg-Neohydrin has been used for tumor diagnostics (Mishkin 1966). The organometallic mercury compound Merbromin (2′,7′-Dibromo-5′-(hydroxymercurio)fluorescein disodium salt, Mercurochrome®) has been used as antiseptic. Because of its mercury content it is no longer sold in Switzerland, France, Germany and the United States.

U.S. Pat. No. 1,672,615 A discloses the antiseptic and antifungal agent Thiomersal (Ethyl(2-mercaptobenzoato-(2-)-O,S) mercurate 1-sodium, Merthiolate®) or thimerosal, respectively, which has been used as a preservative in vaccines, immunoglobulin preparations, skin test antigens, antivenins, ophthalmic and nasal products and tattoo inks. Furthermore, U.S. Pat. No. 1,672,615 A describes a method for the synthesis of water-soluble compounds of alkyl mercuric compounds, which comprises treating a mercuric compound, in which one valence bond is attached to a substituent of other than the sulphur family and the other valence bond is attached to a carbon atom of an alkyl substituent, with an organic compound containing both an acid substituent and a sulfhydryl group directly attached to a carbon atom.

The radiolabeled compound Merisoprol acetate197Hg (hydroxy(2-hydroxypropyl)197mercury, Merprane®) or Merisoprol acetate203Hg, respectively, has been used for diagnosis of renal function.

Disadvantages of the disclosed organometallic mercury compounds are the contamination with203Hg and the toxicity because of the high Hg content.

OBJECT OF THE PRESENT INVENTION

The invention has the object of finding organometallic197(m)Hg compounds with high purity and high specific activity.

CHARACTER OF THE PRESENT INVENTION

The objective of the invention is solved by a197(m)Hg compound according to formula (E)

wherein

Ar is unsubstituted or substituted -aryl or -heteroaryl group,

Y is selected from substituted dithiocarbamates, substituted thiolates, unsubstituted or substituted -aryl or -heteroaryl groups.

In further embodiments the compounds according the invention are selected from compounds according to one of the following formulas (I), (la), (Ib) and (Ic):

wherein each X and W are independently selected from H, unsubstituted or substituted alkyl groups, alkoxy groups with formula —OR1, amide groups with formula —CON(R1)2, carboxy groups with formula —COOR1, aryl or heteroaryl groups,

wherein Y is selected from substituted dithiocarbamates, substituted thiolates, unsubstituted or substituted phenyl and other aryl or heteroaryl groups,

wherein Z is selected from CH, S, N, and O,

wherein Met is selected from Fe, Cr, Mn, Mo, Ru and Rh

197(m)Hg according to the invention is a radionuclide comprising at least one of the two radioactive, γ-emitting nuclear isomers197Hg in the ground state and197(m)Hg in the excited state, wherein m stands for metastable. The nuclear isomer in the excited state,197(m)Hg, emits during its nuclear isomeric transition with a half-life (T1/2) of 23.8 h, a low-energy gamma radiation (Eγ) of 134 keV with 34% probability and conversion electrons with energies between 82 keV and 150 keV. The radioactive Hg isotope197Hg exhibits a half-life (T1/2) of 64.14 h, a low-energy gamma radiation (Eγ) of 77.4 keV with 19% probability and emission of Auger- and conversion electrons.

Preferably, the radionuclide197(m)Hg comprises a molar ratio of197(m)Hg to197Hg of 1:1 to 2:1.

Advantageously, the contamination of the197(m)Hg compound according to formula (I) with other radioactive and non-radioactive Hg isotopes is excluded by the production method according to the invention. Preferably the content of other radioactive Hg isotopes (for example194Hg,195Hg and203Hg) is less than 10−6% of the197(m)Hg content (w/w). Preferably the content of non-radioactive Hg isotopes (196Hg,198Hg,199Hg,200Hg,201Hg,202Hg and204Hg) is below the detection limit of inductively coupled plasma mass spectrometry (ICP-MS) of 1·10−12(w/w).

Preferably the197(m)Hg compound according to formula (I) is produced by the no carrier added (NCA) method as described below.

As used herein, the term “aryl group” refers to unsubstituted or substituted, aromatic hydrocarbon groups. In an embodiment aryl groups are C1 to C18 groups, preferred 5 to 12 groups. In a further embodiment aryl groups are selected from a phenyl group, a tolyl group, a xylyl group and a naphthyl group.

As used herein, the term “heteroaryl group” refers to unsubstituted or substituted, aromatic hydrocarbon groups with at least one heteroatom. Heteroatoms are selected from nitrogen, oxygen, phosphor and sulfur. In an embodiment heteroaryl groups are C1 to C18 groups, preferred 5 to 12 groups. In a further embodiment heteroaryl groups are selected from a furanyl group, pyrrolyl group, thienyl group, oxazolyl group, thiazolyl group, imidazolyl group, pyrazolyl group, pyrimidyl group, pyridazinyl group and indolyl group. In another embodiment heteroaryl groups are selected from 2-Methylbenzfuranyl, 2-Methylbenzothiazyl and 2-Methylthianaphthenyl.

In some embodiments Ar and Y in formula (E) are identical.

In some other embodiments Ar and Y in formula (E) are not identical.

Unsubstituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups according to the invention are hydrocarbon groups without side chains. As used herein, the term “side chains” refers to atoms or atom groups that are attached to a core part of a molecule or the alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups, respectively.

Substituted according to the invention is the replacement of at least one hydrogen atom by an atom or group of atoms on a hydrocarbon compound. The atom or group of atoms is preferably selected from C1 to C15-alkyl, -aryl, -heteroaryl, -alkoxy (—OR2), -carbonyl (—COR2), -amino (—N(R2)2or —NHR2), nitro (—NO2), phosphate groups or halogenides, wherein R2is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups. The carbonyl group can be an aldehyde group (—CHO), a keto group (—COR2), a carboxylic acid group (—COOH), carboxylate ester groups (—COOR1) or an amide (—CON(R2)2).

In an embodiment Xncomprises between 1 and 50 carbon atoms, preferred between 1 and 25 carbon atoms, especially preferred between 1 and 10 carbon atoms.

In a preferred embodiment Xnor X are selected from substituted amide groups.

As used herein, the term “alkyl group” refers to unbranched or branched, unsubstituted or substituted hydrocarbon groups. In an embodiment alkyl groups are C1 to 010 groups, preferred C1 to C3 groups.

In a further embodiment alkyl groups are selected from a methyl group, an ethyl group, a propyl group, an isopropyl group, a pentyl group and a hexyl group.

In a further embodiment Xncomprises at least one heteroatom, preferred two heteroatoms. Heteroatoms are selected from nitrogen, oxygen, phosphor and sulfur.

As used herein, the term “alkoxy group” refers to unbranched or branched, unsubstituted or substituted hydrocarbon groups, wherein at least one oxygen is singular bonded to R1, wherein R1is selected from H, unsubstituted or substituted alkyl, -aryl or -heteroaryl groups. In an embodiment alkoxy groups are C1 to 010 groups, preferred 1 to 3 groups.

In a further embodiment alkoxyl groups are selected from a methoxy group, an ethoxy group and a propoxy group.

As used herein, the term “amide group” refers to unbranched or branched, unsubstituted or substituted hydrocarbon groups, wherein at least one amide group is singular bonded to R1.

As used herein, the term “carboxy group” refers to unbranched or branched, unsubstituted or substituted hydrocarbon groups, wherein at least one carboxy group is singular bonded to R1.

In a preferred embodiment the197(m)Hg compound according to formula (I) is substituted with 1 to 3 Xn, wherein Xnis selected from X1, X2and X3as described above. In a mostly preferred embodiment the197(m)Hg compound according to formula (I) is substituted with one Xnor X, respectively, as shown in formula (I′)

wherein Y is selected from substituted dithiocarbamates, substituted thiolates, unsubstituted or substituted phenyl and other aryl or heteroaryl groups,

In a further embodiment the197(m)Hg compound according to formula (I′) is substituted with X in ortho-, meta- or para-position, preferred in para-position as shown in formula (I″)

wherein Y is selected from substituted dithiocarbamates, substituted thiolates, unsubstituted or substituted phenyl and other aryl or heteroaryl groups,

In a further embodiment Y comprises between 1 and 50, preferred between 1 and 25 carbon atoms, especially preferred between 1 and 10 carbon atoms.

In a further embodiment Y comprises at least one heteroatom, preferred 1 to 6 heteroatoms. Heteroatoms are selected from nitrogen, oxygen, phosphor and sulfur.

Preferably —Y in formula (I), (I′) or (I″) is selected from substituted dithiocarbamates according to formula (II)

wherein R4is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups. The two R3are selected independently.

In a further embodiment R3is selected from unsubstituted or substituted alkyl, alkoxy (—OW), amide (—CON(R4)2), carboxy (—COOR4), aryl or heteroaryl groups.

In a preferred embodiment R3of the substituted dithiocarbamates is selected from substituted amide (—CON(R4)2) or carboxy (—COOR4) groups.

In a further embodiment —Y in formula (I), (I′) or (I″) is selected from substituted thiolates according to formula (III)

In a further embodiment R5of the substituted thiolates is selected from substituted amide (—CON(R6)2) or carboxy (—COOR6) groups.

In a further embodiment —Y is selected from unsubstituted or substituted phenyl groups according to formula (IV)

resulting in a compound according to formula (IV′)

wherein R8is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -aryl or -heteroaryl groups and

X is selected as above.

In a further embodiment R7of the unsubstituted or substituted phenyl groups is selected from substituted amide (—CON(R8)2) or carboxy (—COOR8) groups.

In a further embodiment the197(m)Hg compound according to formula (IV) is substituted with R7in ortho-, meta- or para-position.

In a further embodiment Y is selected from unsubstituted or substituted phenyl groups according to formula (IV), wherein R7and Xnare not identically.

In a further embodiment Y is selected from unsubstituted or substituted phenyl groups according to formula (IV), wherein n is 1 and wherein R7and X are identically resulting in a compound according to formula (V)

In preferred embodiments the compounds according the invention are selected from compounds according to formulas (I), (Ia′), (Ib′) and (Ic′):

wherein each X and W are independently selected from H, unsubstituted or substituted alkyl groups, alkoxy groups with formula —OR1, amide groups with formula —CON(R1)2, carboxy groups with formula —COOR1, aryl or heteroaryl groups,

wherein Y is selected from substituted dithiocarbamates, substituted thiolates, unsubstituted or substituted aryl and heteroaryl groups,

wherein Z is selected from CH, S, N, and O,

wherein Met is selected from Fe, Cr, Mn, Mo, Ru and Rh.

In some embodiments the resulting197(m)Hg-compounds have one of the following formulas:

Preferably, in the197(m)Hg compound according to the invention, both197(m)Hg-substituents are linked by at least one aliphatic and/or aromatic spacer molecule as shown in Formula Ebridge:

wherein the substituents Ar and Y are linked by at least one aliphatic and/or aromatic spacer molecule (symbolized by the line between Ar and Y).

“Aliphatic or aromatic spacer” means a spacer comprising aliphatic or aromatic units (aryl or heteroaryl).

“Aliphatic and aromatic spacer” means a spacer comprising aliphatic as well as aromatic units (aryl or heteroaryl).

The spacer is to be understood as a linker (between Ar and Y in formula (Ebridge) for example). The spacer is an organic unit where all atoms are connected by covalent bonds. The spacer itself does not comprise metal atoms.

Aliphatic units are preferably unsubstituted or substituted alkyl—in some embodiments not comprising alkene units.

Preferably the aliphatic and/or aromatic spacer comprises 4-40 Atoms. In embodiments these atoms comprising 2-5 heteroatoms selected from N, O, S and P. Id est, at least 2 of these 4-40 atoms are heteroatoms.

The aliphatic and/or aromatic spacer molecule is in preferred embodiments an unsubstituted or substituted C6 to C30-alkyl, -alkoxy (—OR9), -amide (—CON(R9)2), -carboxy (—COOR9), -aryl or heteroaryl spacer molecule, preferably a C6-alkyl group or a substituted phenyl group.

“Both197(m)Hg-substituents” in the meaning of the invention, shown exemplarily for formula (Ebridge) means firstly the substituent Y and, secondly, the corresponding aromat Ar which is attached to the Hg via a bond, too.

The “aliphatic and/or aromatic spacer” is more preferably an aliphatic spacer comprising 2-5 heteroatoms, as mentioned above, and comprising 1-3 aryl groups.

A particularly preferred embodiment is that the aliphatic and/or aromatic spacer bears two —CH2—N— groups at the end of each side of the spacer (in the direction: CH2at each end of the spacer).

In embodiments the spacer molecule comprises a reactive group, for example, OH, SH, NH2or COOH (that allows the coupling of further molecules), or a targeting moiety (selected from nucleic acids, antibodies, antibody fragments, peptides, oligonucleotides), alkaloids, carbohydrates, lipids; all of them attached to the spacer via such reactive group.

Most preferably, the aliphatic and/or aromatic spacer has the following formula (VIIIBridge):

wherein R2is H or alkyl, and

R3is selected from: H; a reactive group optionally substituted with a targeting moiety; an alkaloid; a carbohydrate; or a lipid,

the reactive group being selected from OH, SH, NH2and COOH,

and the targeting moiety being selected from a nucleic acid, antibody, antibody fragment, peptide, and oligonucleotide; and

R4and R4′are independently selected from H and Aryl.

Alkyl can be n-butyl especially, or other groups resulting from coupling with Li-organyls.

Preferably, in the197(m)Hg compound according to the invention, both197(m)Hg-substituents are the same, according to formulas (I*bridge), (Ia*bridge), (Ib*bridge) or (Ibridge), (Iabridge), (Ibbridge),

Preferably, the residues Ar and Y in formula (Ebridge) and the aromats shown in (Ibridge), (Iabridge) and (Ibbridge), respectively, are unsubstituted aryl or unsubstituted heteroaryl, i.e. X is H, only the bond to Hg and to the “aliphatic and/or aromatic spacer”.

Preferably, the aliphatic and/or aromatic spacer is connected to both197(m)Hg-substituents in ortho position to the bond to197(m)Hg.

The197(m)Hg compound according to the invention is more preferably a compound of formula (VII)

whereinR2is H or alkyl, andR3is selected from: H; a reactive group optionally substituted with a targeting moiety, an alkaloid, a carbohydrate or a lipid,the reactive group being selected from OH, SH, NH2and COOH,and the targeting moiety being selected from a nucleic acid, antibody, antibody fragment, peptide, and oligonucleotide; andR4and R4′are independently selected from H and Aryl.In one variant of this embodiment, R2is nButyl, R3is OH and both R4and R4′are Phenyl.

Alkyl can be n-butyl especially, or other groups resulting from coupling with Li-organyls.

In further embodiments of the invention both197(m)Hg-substituents are linked by at least one aliphatic and/or aromatic spacer molecule as exemplarily shown in formulas (Ibridge), (Iabridge), (Ibbridge) or (Icbridge).

wherein each X and W are independently selected from H, unsubstituted or substituted alkyl groups, alkoxy groups with formula —OR1, amide groups with formula —CON(R1)2, carboxy groups with formula —COOR1, aryl or heteroaryl groups,

wherein Y is selected from substituted dithiocarbamates, substituted thiolates, unsubstituted or substituted aryl or heteroaryl groups,

wherein Z is selected from CH, S, N, and O,

wherein Met is selected from Fe, Cr, Mn, Mo, Ru and Rh,

preferably as shown in formulas (Ibridge), (Iabridge) and (Ibbridge).

In a preferred embodiment the aliphatic and/or aromatic spacer molecule is located in ortho-position or meta-position relating to the position of the197(m)Hg-moiety, at the aryl or heteroaryl groups of formulas (Ibridge), (Iabridge), (Ibbridge) or (Icbridge).

In a preferred embodiment the phenyl groups of the197(m)Hg compound according to the invention are linked by at least one aliphatic and/or aromatic spacer molecule as shown in formula (VI)

In an embodiment the197(m)Hg compounds according to the invention further comprise at least one amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue and/or aliphatic spacer.

In a further embodiment Xnand/or Y further comprise at least one amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue and/or aliphatic spacer. In an embodiment the aliphatic spacer is selected from polyethylene glycol.

In an embodiment Xnand/or Y comprises one aliphatic spacer and one amino acid, peptide, protein, antibody, oligonucleotide or alkaloid residue.

Advantageously, the197(m)Hg compounds according to the invention exhibit high purity and high specific activity.

As used herein, the term “purity” refers to the amount of197(m)Hg compounds according to the invention based on the amount of substance.

As used herein, the term “specific activity” refers to the amount of radioactive decay per time interval (1 decay per second=1 Becquerel (Bq)) based on the molar amount of substance. The specific activity of the197(m)Hg compound according to the invention is based on the molar amount of the197(m)Hg compound. The specific activity can be determined for example by inductively coupled plasma mass spectrometry (ICP-MS).

In an embodiment the197(m)Hg compounds according to the invention have a specific activity of at least 100 GBq/μmol based on the molar amount of the197(m)Hg compound, i.e. of mercury, preferred 100 to 1.000 GBq/μmol based on the molar amount of the197(m)Hg compound.

In an embodiment the197(m)Hg compounds of the invention can be used in nuclear medical diagnostics and endoradionuclide therapy (theranostic).

In a further embodiment the197(m)Hg compounds of the invention can be used in the treatment of cancer.

In a further embodiment the197(m)Hg compounds of the invention can be used for the manufacture of a medicament for endoradionuclide therapy.

In a further embodiment the197(m)Hg compounds of the invention can be used for the manufacture of a medicament for the treatment of cancer.

In a further embodiment the197(m)Hg compounds of the invention can be used as an active ingredient for the preparation of a pharmaceutical composition.

The present invention further comprises a pharmaceutical composition comprising a197(m)Hg compound of the invention.

The present invention further comprises a method for the production of the197(m)Hg compounds according to the invention comprising the steps:a) Provision of an organic precursor compound,b) Synthesis of no carrier added (NCA)197(m)Hg,c) Radiolabeling of the organic precursor compound with the no carrier added (NCA)197(m)Hg by electrophilic substitution.

Advantageously, the method for the production of197(m)Hg compounds according to the invention is fast and is carried out under moderate conditions. As used herein, the term “fast” refers to periods of a few minutes to a few hours, preferred 5 min to 2 h. As used herein, the term “moderate conditions” refers to moderate temperatures of 25 to 70° C. Advantageously, compounds with the radioactive Hg isotopes197Hg and197(m)Hg can be synthesized by the method according to the invention and administered to patients for the use in nuclear medical diagnostics and endoradionuclide therapy (theranostic), preferred in the treatment of cancer, before the half-life (T1/2(197Hg)=64.14 h, T1/2(197mHg)=23.8 h) of the radioactive Hg isotopes has passed. Furthermore advantageously, temperature-sensitive molecules, for example peptides, proteins, nucleic acids or antibodies, are preserved under the moderate conditions.

In an embodiment the method for the production of197(m)Hg compounds according to the invention is carried out in the order of the steps a), b) and c).

In a further embodiment the method for the production of197(m)Hg compounds according to the invention is carried out in the order of the steps b), a) and c).

As used herein, the term “organic precursor compound” refers to a hydrocarbon compound comprising at least one heteroatom.

In an embodiment the organic precursor compound provided in step a) is an organotin precursor compound, a boron precursor compound or a silicon precursor compound according to formulas (Iprec), (Iaprec), (Ibprec) or (Icprec)

wherein each X and each W are independently selected from H, unsubstituted or substituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups, wherein R1is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups,

Z is selected from CH, S, N, and O,

M is Sn, B or Si;

wherein Met is selected from Fe, Cr, Mn, Mo, Ru and Rh,

i is 2 or 3.

In a preferred embodiment the organic precursor compound is an organotin precursor compound, a boron precursor compound or a silicon precursor compound, wherein n and o are 1, according to formulas (Iprec′), (Iaprec′), (Ibprec′), or (Icprec′).

wherein each X and each W are independently selected from H, unsubstituted or substituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups,

Z is selected from CH, S, N, and O,

M is Sn, B or Si,

wherein Met is selected from Fe, Cr, Mn, Mo, Ru and Rh,

i is 2 or 3.

In an embodiment the organic precursor compound according to formulas (Iprec′), (Iaprec′), (Ibprec′), or (Icprec′) is substituted with X in ortho-, meta- or para-position, compared to substituent M(R10)i.

In a preferred embodiment the organic precursor compound is a tin precursor compound, especially preferred a trialkyl-tin precursor compound. Trialkyl-tin precursor compounds are selected from tri-n-butyl-tin precursor compounds or trimethyl-tin precursor compounds (according to the following formulas):

In a further embodiment the organic precursor compound is synthesized by catalytic reaction of the halogen compound. In a preferred embodiment the organic precursor compound is synthesized by catalytic reaction of the halogen compound with an alkyl-tin compound, an alkyl-boron compound or an alkyl-silicon compound.

In some embodiments, the invention provides an organic precursor compound according to formula (Ebridge-prec):

wherein both Ar and Y are linked by at least one aliphatic and/or aromatic spacer molecule, wherein Ar is unsubstituted or substituted -aryl or -heteroaryl group, and Y is selected from unsubstituted or substituted -aryl and -heteroaryl groups;M is Sn, B or Si;R10is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups, andi is 2 or 3.The aliphatic and/or aromatic spacer molecule, Ar as well as Y are defined and preferably selected as described above for197(m)Hg-compound.

In a preferred embodiment of the method above, in step a) of the method an organic precursor compound according to formula (Ebridge-prec) is provided.

Preferably the organic precursor compound according to the invention is one according to formulas (Ibridge-prec), (Iabridge-prec) or (Ibbridge-prec):

with the definitions as above, andwherein each X is independently selected from H, unsubstituted or substituted alkyl groups, alkoxy groups with formula —OR1, amide groups with formula —CON(R1)2, carboxy groups with formula —COOR1, aryl or heteroaryl groups,wherein R1is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups.

In a further embodiment the synthesis of NCA197(m)Hg according to step b) is carried out by irradiation of gold (Au) with a cyclotron. As used herein, the term “no carrier added (NCA)” refers to preparation of a radioactive isotope without the addition of stable isotopes of the element in question.

In a further embodiment the NCA197(m)Hg synthesised according to step b) is NCA197(m)HgCl2.

In a further embodiment the synthesis of NCA197(m)Hg according to step b) is followed by purification of the NCA197(m)Hg by liquid-liquid extraction or solid-phase extraction.

In a further embodiment the radiolabeling of the organic precursor compound according to step c) is carried out by addition of NCA197(m)HgCl2to the organic precursor compound.

In a further embodiment the radiolabeling of the organic precursor compound according to step c) is carried out by addition of the NCA197(m)Hg to the organic precursor compound in a molar ratio of 1:10 to 1:1.000 (n/n).

In a further embodiment the radiolabeling of the organic precursor compound according to step c) is carried out at a pH value between pH 1.0 and 7.0.

In a further embodiment the radiolabeling of the organic precursor compound according to step c) is carried out at a pH value between pH 6.0 and 7.0 to form symmetric197(m)Hg compounds.

As used herein, the term “symmetric197(m)Hg compounds” refers to197(m)Hg compounds, wherein197(m)Hg exhibits two identical binding partners. Symmetric197(m)Hg compounds are197(m)Hg compounds according to formula (V) and (VI).

In a further embodiment the radiolabeling of the organic precursor compound according to step c) is carried out at a pH value between pH 1.0 and 5.0 to form asymmetric197(m)Hg compounds.

As used herein, the term “asymmetric197(m)Hg compounds” refers to197(m)Hg compounds, wherein197(m)Hg exhibits two different binding partners. Asymmetric197(m)Hg compounds are197(m)Hg compounds of the invention, except197(m)Hg compounds according to formula (V) and (VI).

In a further embodiment the formed asymmetric197(m)Hg compounds are added to dithiocarbamate ligands to form197(m)Hg compounds according to formula (II).

In a further embodiment the radiolabeling of the organic precursor compound according to step c) is carried out by addition of dimethyl sulfoxide (DMSO). Advantageously, DMSO increases the solubility of the organic precursor compound.

In a further embodiment the radiolabeling of the organic precursor compound according to step c) is followed by reaction of activated ester groups by ester hydrolysis, reaction with amino groups or reaction with hydroxyl groups of an amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue and/or aliphatic spacer.

In a further embodiment ester hydrolysis is carried out with sodium hydroxide solution.

In a further embodiment reaction of activated ester groups with amino groups of an amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue and/or aliphatic spacer is carried out at a pH value between pH 8.0 and 9.0.

The present invention further comprises an organic precursor compound according to formulas (Iaprec), (Ibprec) or (Icprec) for the use in the production of the197(m)Hg compounds according to the invention.

The present invention further comprises an organic precursor compound according to formulas (Iaprec), (Ibprec) or (Icprec) for the use in the method according to the invention.

The present invention further comprises a method for nuclear medical diagnostics and endoradionuclide therapy (theranostics) of cancer with the197(m)Hg compounds according to the invention.

The method for nuclear medical diagnostics and endoradionuclide therapy (theranostics) includes the step of administering to a subject in need thereof, a pharmaceutical composition containing a therapeutically effective amount of197(m)Hg compounds according to the invention.

In an embodiment the method for treatment further comprises a nuclear medical diagnostic of the therapeutic efficacy of the197(m)Hg compounds according to the invention.

A pharmaceutical composition containing the197(m)Hg compounds according to the invention typically contains a pharmaceutically acceptable carrier, such as saline. The dose of the197(m)Hg compounds according to the invention is preferably 1 GBq to 5 GBq. The subject may be a mammal, such as a human.

The dose of 1 GBq to 5 GBq of the197(m)Hg compounds according to the invention with preferably a specific activity of at least 100 GBq/μmol based on the amount of mercury refers to a dose of 10 nmol to 50 nmol of mercury or 2 μg to 10 μg of mercury, respectively. Mostly preferred the197(m)Hg compounds according to the invention has a maximal specific activity of 1,000 GBq/μmol, which refers to a dose of 1 nmol to 5 nmol of mercury or 0.2 μg to 1 μg of mercury, respectively. Advantageously, these doses of mercury are in the same order of magnitude as the estimated daily Hg intake of the European and North American general population or clearly below and therefore do not lead to toxic concentrations in patients (Clarkson and Magos 2006).

Although the invention describes various dosages, it will be understood by one skilled in the art that the specific dose level and frequency of dosage for any particular subject in need of treatment may be varied and will depend upon a variety of factors. These factors include the metabolic stability of the197(m)Hg compounds according to the invention and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. Generally, however, dosage will approximate that which is typical for known methods of administration of the specific compound. Thus, a typical dosage of the197(m)Hg compounds according to the invention will be about 5 to 50 MBq/kg.

The pharmaceutical compositions and formulations containing the197(m)Hg compounds according to the invention can be administered systemically. As used herein, “systemic administration” or “administered systemically” refers to compositions or formulations that are introduced into the blood stream of a subject, and travel throughout the body of the subject to reach the part of the subject's body in need of treatment at an effective dose before being degraded by metabolism and excreted. Systemic administration of compositions or formulations can be achieved by intravenously injection.

Pharmaceutical compositions containing the197(m)Hg compounds according to the invention are prepared for administration and/or storage by mixing the197(m)Hg compounds according to the invention, after achieving the desired degree of purity, with pharmaceutically and/or physiologically acceptable carriers, auxiliary substances or stabilizers (Remington's Pharmaceutical Sciences) in the form of a lyophilisate or aqueous solutions. The term “pharmaceutically acceptable” or “physiologically acceptable,” when used in reference to a carrier, is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. Acceptable carriers, auxiliary substances or stabilizers are not toxic for the recipient at the dosages and concentrations employed; they include buffers such as phosphate, citrate, tris or sodium acetate and other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (less than approximately 10 residues), proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, leucine or lysine; monosaccharides, disaccharides and other carbohydrates, for example glucose, sucrose, mannose, lactose, citrate, trehalose, maltodextrin or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium, and/or non-ionic surface-active substances such as Tween, Pluronics or polyethylene glycol (PEG).

Such pharmaceutical compositions may further contain one or more diluents, fillers, binders, and other excipients, depending on the administration mode and dosage form contemplated. Examples of therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, saccharose, alcohols, glycerin and the like. Various preservatives, emulsifiers, dispersants, flavorants, wetting agents, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like can also be added, as required to assist in the stabilization of the formulation or to assist in increasing bioavailability of the active ingredient(s). The197(m)Hg compounds according to the invention can be administered alone, or in various combinations, and in combination with other therapeutic agents. The197(m)Hg compounds used in the invention are normally stored in solution.

Preferably the197(m)Hg compound according to the invention is the compound of formula (3*)

The corresponding organic precursor compound preferably is compound (2)

Advantageously, compound (3*) is highly in-vivo. This is shown as there isn't any observable protein interaction by human serum testing. Secondly, the compound leads to good organ clearance demonstrated by biodistribution and SPECT studies in rats, in particular there is no retention in the kidneys typical of unstable mercury compounds.

Still, it has functionality allowing its binding to a tumor-targeting carrier, namely via the OH-group, with known methods.

Furthermore, (3*) showed high chemical stability in tests with an excess of sulfur-containing competitors (glutathione, tris(2-mercaptoethyl)ammonium oxalate and sodium sulfide).

In a further embodiment the recently described embodiments can be combined.

All preferred embodiments of the invention count for the197(m)Hg-compound and for the corresponding organic precursor compound as well.

GENERAL SYNTHETIC TECHNIQUES

All Chemicals were used without further purification and in the highest degree of purity.

Sodium hydroxide in suprapur quality was purchased from Merck (Darmstadt, Germany). Methyl isobutyl ketone (MIBK) was purchased from Sigma-Aldrich (St. Louis, USA). The routine activity measurement was performed with an Isomed 2000 from MED (Nuklear-Medizintechnik Dresden GmbH, Dresden, Germany) calibrated by γ-ray spectroscopy measurements after decaying197(m)Hg. ICP-MS measurements were carried out on an ELAN 9000 (PerkinElmer SCIEX, Waltham, USA).

For γ-ray spectroscopy measurements a reverse electrode HPGe detector (CANBERRA GR2018, 19.6% rel. efficiency) in a low-background Pb shielding was used with the sample at 10 cm distance from the detector end cap. It was operated with the software InterWinner version 7.1. The system was calibrated using a mixed standard solution (57Co, 85Sr, 88Y, 60Co, 109Cd, 113Sn, 137Cs, 139Ce, 203Hg, 241Am) with a volume of 0.38 mL in the tip of a 1.5 mL Eppendorf vial. The energy depending detector efficiency was calculated from these calibration points using the algorithms of the spectroscopy software. The samples were measured in similar geometry, but smaller volume of 1-10 μl in the tip of a 1.5 mL Eppendorf vial thus, no further corrections were necessary with except of decay correction. Pile-up effects were observed, especially at higher activities. Nevertheless, no corrections are made, because the effects are less than the simple standard deviation and thus negligible. For the determination of Hg-activities only the γ-ray lines >100 keV have been used, in particular for the isomer 197mHg only the lines ˜134 keV and ˜165 keV of the isomeric transition and for the isomer 197Hg only the lines ˜191 keV and ˜269 keV are discussed in the activity calculation.

NMR and IR Spectroscopy

1H and13C NMR spectra were recorded with a Varian Inova-400 spectrometer. The chemical shifts were reported relative to the standard tetramethylsilane (TMS). IR spectra were measured with a Fisher Scientific Nicolet iS5 FTIR spectrometer.

Thin Layer Chromatography (TLC)

Thin layer chromatography was performed using RP18 plates (Merck), developed in a 1:1 mixture H2O with 0.1% trifluoroacetic acid (TFA) (A) and CH3CN with 0.1% TFA (B) and analyzed with a Raytest Linearanalyser RITA.

Radio-TLC is the detection of radioactive species separated by TLC with radiation detector to determine the radiochemical purity or to quantify the radioactive species.

The radiochemical yield is the yield of the radionuclide and was calculated by the specific activity of the197(m)Hg compound divided by the specific activity of the no carrier added (NCA)197(m)Hg.

Radiochemical purity was determined by radio-HPLC. All HPLC runs are performed under the same conditions with the same HPLC-equipment. Column: Zorbax C18 column with inner diameter of 8 mm. Mobile phase: H2O with 0.1% TFA (A) and CH3CN with 0.1% TFA (B). Flow rate: 3 mL/min. HPLC gradient of B phase: in 0 to 20 min from 45% to 80%, in 20 to 25 min from 80% to 100%.

For mass spectrometry a QuadroLC by Micromass with electrospray ionisation (ESI) mode and a Bruker MALDI-TOF MS instrument (MALDI) were used.

1. Synthesis of an Organic Precursor Compound

3-iodobenzylamine hydrochloride salt (4 g, 14.84 mmol) was dissolved in chloroform (100 ml) in a 250 ml round-bottomed flask. To this was added triethylamine (10.3 ml, 0.074 mol) followed by isophthaloyl chloride (1.51 g, 7.42 mmol). The flask was fitted with a CaCl2) drying tube and the colourless solution was left to stir at room temperature overnight. The reaction was monitored by TLC using 19:1 dichloromethane (DCM)/methanol (MeOH). The reaction mixture was washed with 3:1 water/saturated NaHCO3(aq.)(3×50 ml), then with 0.1 M HCl(aq.)(3×50 ml), then with deionized water (2×30 ml). The product is mostly insoluble in chloroform and precipitates during the aqueous washes, thus further dilution with chloroform helps separation. The product was purified by simple recrystallization of cooling the chloroform. Impurities dissolved in the solvent were decanted. This process was repeated to increase yield. The product was washed lightly with cold chloroform and after drying left a white powder (1.02 g, 92% yield).

N1,N3-bis(3-iodobenzyl)isophthalamide (0.97 g, 1.63 mmol) was dissolved in 1,4-dioxane (20 ml) in a 50 ml 3-necked round-bottomed flask. A glass bubbler allowed argon to bubble through the solution with a coiled water condenser attached to the top along with a bubble counter to monitor argon flow. A catalytic amount of tetrakis(triphenylphosphine)palladium(0) (20.4 mg, 16.3 μmol) orange crystals were added forming a clear pale yellow solution. This was followed by an excess of hexamethylditin (3.16 ml, 15.26 mmol). Rinsing of sample phials and addition funnel brought the total solvent volume to 30 ml. The reaction mixture was heated by an oil bath (125° C.) and stirred for 8 h. The reaction was monitored by TLC using 1:1 ethanol (EtOH)/n-hexane. The reaction mixture turned a dark orange with a cloudy precipitate. This was filtered to remove most of the brown precipitate. The solvent was removed by evaporation and the product purified by flash column chromatography using EtOH/n-hexane. Drying yielded a white powder (0.164 g, 15% yield).

2. Production of No-Carrier-Added197(m)Hg

The irradiations were performed at a Cyclone 18/9 cyclotron (IBA, Louvain la Neuve, Belgium, 18 MeV protons) located at Dresden-Rossendorf. A 1.0 mm aluminum foil (high purity aluminum, 99.999%) from Goodfellow (Huntingdon, England) was used as vacuum window. As target material massive high purity gold disks (23 mm diameter, 2 mm thickness, N5 purity 99.999%) were purchased from ESPI (Ashland, USA). Alternative gold targets consisted of a gold foil (12.5×12.5 mm, 0.25 mm thickness, 99.99+%) or a small gold disk (10 mm diameter, 0.125 mm thickness, 99.99+%, Pt content: 45±5 ppm quantified per ICP-MS) between an aluminum disk (22 mm diameter, 1 mm thickness, 99.0%, hard) and an aluminum lid (23 mm diameter, 99.0%, hard) purchased from Goodfellow (Huntingdon, England). Hydrochloric acid (30%) and nitric acid (65%) were purchased from Roth (Karlsruhe, Germany) in Rotipuran® Ultra quality. Deionized water with >18 MΩcm resistivity was prepared by a Milli-Q® system (Millipore, Molsheim, France). LN resin was purchased from Triskem International (Bruz, France). The gold target was irradiated for 120 min with a 25 μA current of 10 MeV protons resulting in 200 MBq of197(m)Hg. The irradiated gold foil was dissolved in 700 μl of aqua regia (freshly prepared 1 h before EOB from 525 μl 30% HCl+175 μl 65% HNO3) at room temperature. The gold disk was completely dissolved after 50 to 60 min. The column preparation was carried out directly before use by loading 3.6 g LN resin slurried with 10 ml of 6 M HCl onto the column and rinsing with additional 30 ml of 6 M HCl. After dilution of the 700 μl product solution with 300 μl 6 M HCl, this mixture was loaded onto the column and eluted with 6 M HCl in 1 ml aliquots.

FIG. 1shows the fractionated elution of197(m)Hg mercury chloride in 6 M HCl (two major fractions 7+8 and two minor fractions 9+10) and198Au+196Au containing chloroauric acid in 0.1 M HCl (fractions 13-22).

3. Radiolabeling of the Organic Precursor Compound with the No Carrier Added (NCA)197(m)Hg by Electrophilic Substitution

General Synthetic Procedure for Synthesis of DiphenylnatMercury Compounds (Reference) Based on Sn-Precursors:

A solution of one equivalent mercury (II)-chloride was added to a solution of two equivalents tin-precursor in acetonitrile. The immediately starting precipitation of the product was completed by addition of ice cooled diethyl ether after 2 h mixing at room temperature. Centrifugation followed by washing the residue with cold diethyl ether results in a colorless microcrystalline product.

A solution of one equivalent mercury (II)-chloride (5.5 mg, 20 μmol) in 1.5 ml acetonitrile was added to a solution of two equivalents tin-precursor N-succinimidyl-4-(tri-n-butylstannyl)benzoate (21 mg, 41 μmol) in 1.5 ml acetonitrile. The immediately starting precipitation of the product was completed by addition of ice cooled diethyl ether after 2 h mixing at room temperature. Centrifugation followed by washing the residue with cold diethyl ether results in a colorless microcrystalline product.

General Synthetic Procedure for Synthesis of Radiolabeled Diphenyl-Mercury Species—Based on Sn-Precursors

The197(m)Hg chloride stock solution in 0.2 M HCl is adjusted to pH 6 by adding 100 μl 0.2 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer and 5-10 μl 1 M NaOH. A solution of 1-10 μg trialkyltin precursor in 50-100 μl dimethyl sulfoxide (DMSO) is added to this buffered197(m)Hg chloride solution and mixed at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).

The197(m)Hg chloride solution in 0.2 M HCl is adjusted to pH 6 by adding 100 μl 0.2 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer and 5-10 μl 1 M NaOH. A solution of 10 μg (20 nmol) N-succinimidyl-4-(tri-n-butylstannyl)benzoate in 100 μl DMSO is added to 110 μl of this buffered197(m)Hg chloride solution (45 MBq [197(m)Hg] mercury) and mixed at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (ACN/H2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).

General Synthetic Procedure for Synthesis of Diaryl/HeteroarylnatMercury Compounds (HPLC Reference)-Based on B-Precursors

A mixture of one equivalent mercury (II)-acetate (5 μmol), ten equivalents boronic acid (50 μmol) and ten equivalents cesium carbonate (50 μmol) in 1 ml propane-2-ol was tempered at 50° C. for 20 h. After cooling and drying the mixture by rotary evaporation the product was extracted from the residue with toluene or THF purified by HPLC and identified by mass spectrometry.

A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 2-thienylboronic acid (6.4 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.

A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 5-(Dihydroxyboryl)-2-thiophenecarboxylic acid (8.5 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.

A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents ferroceneboronic acid (11.5 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.

A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 5-(dihydroxyboryl)-3-pyridinecarboxylic acid (8.3 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.

A solution of one equivalent phenylmercury acetate (1.7 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 5-(dihydroxyboryl)-2-thiophenecarboxylic acid (8.5 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.

General Synthetic Procedure for Synthesis of Radiolabeled Diaryl/Heteroaryl−Mercury Species

Based on B-Precursors

A solution of 10-100 μg aryl boronic acid precursor in 50-100 μl ethanol is added to the intended amount197(m)Hg acetate solution in 0.2 M sodium acetate. The pH of the mixture is then adjusted to pH 8 by adding 100 μl 0.2 M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer and shaken at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).

This heteroleptic diaryl mercury compound is accessible in a two-step procedure (analogous to the asymmetric phenylmercury dithiocarbamate derivatives (see next section):

The197(m)Hg chloride stock solution in 0.2 M HCl is diluted by adding 100 μl water and 100 μl ethanol to improve the solubility of the tin precursor and the lipophilic intermediate. A solution of 10 μg trimethylstannyl benzene precursor in 50 μl dimethyl sulfoxide (DMSO) is added to this acidic197(m)Hg chloride solution and mixed at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).

Step 2: Reaction of the197(m)Hg Phenylmercury Chloride with the Aryl Boronic Acid

A solution of 50 μg 5-carboxy-2-thienylboronic acid in 50 μl ethanol is added together with 100 μl 0.2 M sodium acetate to the197(m)Hg phenylmercury chloride. The pH of the mixture is then adjusted to pH 8 by adding 100 μl 0.2 M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer and shaken at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).

Synthesis of Asymmetric Radiolabeled Aryl-Mercury-Dithiocarbamate Derivatives

2 μg of the tin precursor N-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(tributylstannyl)benzamide (K08-15) dissolved in 20 μl DMSO was added into 50 μl 0.1 M HCl solution containing 45.5 MBq [197(m)Hg]HgCl2. The reaction mixture was shaken overnight at 25° C. (>12 h). Acidic environment is needed to avoid the formation of symmetric diphenyl mercury species. Excess of organotin precursors were decomposed slowly in acid environment.

The pH of the phenyl mercury chloride derivatives (step 1) was adjusted to pH 6, adding about 200 μl 0.2 M MES buffer (pH 6.0 to 6.2) and about 10 μl 0.2 M NaOH, before the dithiocarbamate ligand is added. Then 20 μg dithiocarbamate (cw04) containing 50 μl 0.2 M MES buffer (pH 6.0 to 6.2) were added into mixture quickly. Then, the reaction mixture was shaken at 50° C. for 60 min.

Radiochemical purity was determined by radio-HPLC (seeFIG. 2). The non-radioactive reference substance of dithiocarbamate arylmercury derivative was used to confirm the labeling product either.FIG. 2shows a) Radiochromatogram of Phenyl-197(m)Hg-dithiocarbamate and b) UV-chromatogram of non-radioactive Phenyl-Hg-dithiocarbamate (reference).

To a solution of 23 mg (36 μmol) Bis(4-(N-succinimidyl)benzoate)mercury(II) in 2 ml dimethylformamide (DMF) 2.88 μl 2.5 N NaOH (72 μmol) and 1 ml water were added. After mixing 2 h at 50° C. the completion of the reaction was confirmed by TLC control (DCM/MeOH 50:1 (v/v), DC silica gel 60 F254). The pH was adjusted to pH 3 by addition of acetic acid then the solvent was removed by rotary evaporation and residue redissolved in 2 ml DMF. The product was precipitated by addition of 20 ml cold diethyl ether, filtrated and dried under vacuum, resulting in a white solid.

The solution of [197(m)Hg] Bis(4-(N-succinimidyl)benzoate)mercury (II) is adjusted to pH 9 by adding 10 μl 1 M NaOH and mixed for 1 h at 50° C. The completion of the reaction is confirmed by TLC control (ACN/H2O 90:10 (v/v) with 0.1 vol-% TFA, ITLC-SG and RP18 material). Finally, the pH is adjusted to pH 6-7 by addition of 10 μl 1 M HCl.

5. Synthesis of the [197(m)Hg] Bis(4-carboxyphenyl)mercury (II)-mAb Cetuximab (C225) Conjugate by Prelabeling with the Labeled Active Ester

The solution of [197(m)Hg] Bis(4-(N-succinimidyl)benzoate)mercury (II) is added to a solution of 1 mg size-exclusion chromatography (SEC) purified C225 antibody in HEPES buffer at pH 8. After mixing the pH is adjusted to pH 8.5. After 1 h at 37° C. the progress of the reaction is confirmed by TLC control. (ACN/H2O 90:10 (v/v) with 0.1 vol-% TFA, ITLC-SG and RP18 material). Unreacted active ester residues were quenched by adding 10 μl 1 M tris(hydroxymethyl)aminomethane (TRIS) solution and separated using a PD10 desalting column.

6. Synthesis and Stability of Compound (3*)

The synthesis is schematically shown in the following scheme:

Compound (3*) was characterized by UV (FIG. 3), HPLC, MS and NMR.

In vivo stability of (3*) was tested (FIG. 4) as via incubation with human serum as per the procedure of Zarschler et al. (Zarschler, K.; Kubeil, M.; Stephan, H. Establishment of Two Complementary in Vitro Assays for Radiocopper Complexes Achieving Reliable and Comparable Evaluation of in Vivo Stability. RSC Adv. 2014, 4 (20), 10157-10164).

The results (FIG. 4) show that, unlike with 197(m)Hg-radiolabeled EDTA used as reference, neither 197(m)Hg release (demetallation) nor binding to serum proteins is detectable for 3*. The only radioactive species detected matches with the mass of 3* highlighting its remarkable stability under these conditions. In contrast, a range of protein bands of different sizes are visible when197(m)Hg-radiolabeled EDTA was incubated with human serum due to substantial decomplexation and transchelation.

To test the actual in vivo stability of 3*, a biodistribution was performed on healthy rats and the results (FIG. 5) show good renal clearance, by normal micturition during a 24 h period, and no indication of demetallation and retention in the kidneys as observed with mercury salts.

Preparation of 3,7-bis(2-bromobenzyl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one (1)

Preparation of 9-butyl-1,5-diphenyl-3,7-bis(2-(trimethylstannyl)benzyl)-3,7-diazabicyclo[3.3.1]nonan-9-ol (2)

Preparation of 9-butyl-8,10-diphenyl-6,10:8,12-dimethanodibenzo[c,f][1,9]diaza[5]mercuracyclotetradecan-9-ol (3)

The radionuclide was prepared by the bombardment of high purity 197Au target (99.99+%, 10 mm diameter, 0.125 mm thickness, Safina, Czech Republic) with a deuteron beam of the cyclotron U-120M in the Nuclear Physics Institute of the CAS, Czech Republic. The irradiations were per-formed using 15.8 MeV deuterons at the beam current of 10 μA for 4 h. It resulted in ˜0.58 GBq of 197gHg and ˜1.14 GBq of 197mHg at EOB, respectively. After arrival at HZDR, Germany, the irradiated targets were dissolved in aqua regia (700 μl), prepared from 30% HCl(aq) (525 μl) and 65% HNO3(aq) (175 μl) (purity Trace-Select, Sigma-Aldrich), and diluted with 6M HCl(aq) (300 μl). The resulting solution had a total activity of ˜0.9 GBq. 0.5 μl (˜1.57 MBq) was removed as a reference substance and the rest of the solution was carefully loaded onto a prepared column filled with 3.6 g of LN resin (LN-B100-A, 100-150 μm, TRISKEM, France) that had been soaked for 15 min in 6M HCl(aq), rinsed slowly with 6M HCl(aq) (30 ml), capped with a frit, overlayered with ca. 1 cm of sand and finally rinsed with 6M HCl(aq) (80 ml). After loading the target solution, the column was slowly washed with 6M HCl(aq) (6×1 ml) fractions, minor activity being detected from the 5th fraction, the fraction volume was reduced (6×0.5 ml). Most of the activity was eluted in the 9th-11th fractions.

Radiolabeling Procedure for Stability Tests:

After pH-adjustment of the hydrochloric acid solution of [197(m)Hg]HgCl2(aq) (˜55 MBq, 20 μl, pH 1), by addition of 0.5 M HEPES buffer (pH 8, 200 μl), EtOH (200 μl), 6 M NaOH(aq) (11 μl), and 1 M NaOH(aq) (2 μl), a 1 mg/ml acetonitrile solution of 2 (12 μl, 14 nmol) was added. This solution (pH 6, 445 μl) was mixed at 50° C. for 1 h. The radiochemical yield of 3* was determined by radio-TLC (iTLC ACN+0.1% TFA, RP-18 TLC 9:1 ACN: H2O+0.1% TFA) as >95%. Purification and solvent change were carried out with a C8 cartridge (500 mg). After washing with water the major product was eluted with 7:3 EtOH:H2O from the cartridge. The last 2 fractions contained ˜16 MBq and ˜10 MBq respectively. The ˜16 MBq fraction had 3×200 μl extracted (˜4 MBq each). These fractions then had 1 competitor added each (1 mg/ml, 10 μl): tris(2-mercaptoethyl)ammonium oxalate, glutathione and Na2S. The mixtures were left at rt and checked by radio-TLC after 5 min, 1 h and 2 d, the only degradation observed was ˜4% after 2 d in the Na2S mixture. The ˜10 MBq fraction was divided into 2×500 μl. The first lot was used to test the stability of 3* in the highly aqueous solvent system necessary for biodistribution studies, this was diluted from 70% EtOH(aq) to ˜10% EtOH(aq) with brine (3.5 ml). Transferal to a fresh vial showed negligible loss in activity and radio-TLC of the solution showed good stability. The other 500 μl lot was used to test the volatility of 3*: firstly the sample was diluted with 70% EtOH(aq) (500 μl) and the vial heated to 50° C. whilst a stream of dry nitrogen was blown onto the 1 ml solution for 1 h until the solution volume had been reduced to ˜350 μl. Transferal to a fresh vial showed negligible loss in activity and measurement of the remaining solution showed no observable loss by evaporation.

Determination of Distribution Coefficient of (3*):

Shake flask method: Into a 10 ml glass vial was added n-octanol (500 μl), 0.05 M HEPES buffer solution (pH 7.4, 475 μl) and a 1:1 EtOH:H2O solution of 3* (25 μl). The vial was shaken for 30 s, then 400 μl extracted from each phase and centrifuged separately. 2×100 μl was taken from each phase and the intensity of radioactivity was measured by a gamma counter and averaged.

Human Serum Stability Assay:

Human serum “off the clot” (5 ml) stored at −20° C. was slowly thawed on ice and filtered using syringe filters with a pore size of 0.2 μm. Two aliquots of filtered serum (2×220 μl) were mixed with 1 M HEPES/NaOH buffer (pH 7.4, 2×45 μl). Separately, 2×200 μl solution (1:1 EtOH:H2O, pH 6) of 3* (˜4 MBq) and 197(m)HgCl2/EDTA (˜5 MBq, 10 μg EDTA) had 1 M HEPES/NaOH buffer (pH 8.0, 2×20 μl) added to increase solution pH to 7.4. Then 135 μl of each 197(m)Hg-radiolabeled sample was added to one of the previously prepared serum/buffer solutions (265 μl) and incubated for 1 h at 37° C. 50 μl aliquots were then taken and mixed with 50 μl of 2× Laemmli sample buffer (Bio-Rad Laboratories), N.B. no reducing agent was added and the samples were not heated. The mixtures were then analyzed by non-reducing SDS-PAGE with acrylamide concentrations of 5% in the stacking gel and 20% in the resolving gel. 2 μl of each sample were loaded into each gel well. The SDS-PAGE was run at r.t. and 80 V until the dye front reached the resolving gel and then increased to 140-160 V. After electrophoresis, the gel was washed for 1 min with H2O and then exposed to a high-resolution phosphor imaging plate (GE Healthcare) for 10 min and the exposed plate scanned (Amersham Typhoon 5 Scanner, GE Healthcare) to measure an autoradiograph. The gel was then stained with PageBlue protein staining solution (Thermo Fisher Scientific, Coomassie G-250).

CITED NON-PATENT LITERATURE