Patent Application
20210228748
There has been a continuing need for effective radioisotopes in nuclear medical diagnostics and endoradionuclide therapy (theranostics).
The interest in the mercury isotope 197(m)Hg was awakened primarily by the decay characteristics of both nuclear isomers, like convenient half-life 197(m)Hg (T1/2=23.8 h, Eγ 134 keV, 34%) and 197Hg (T1/2=64.14 h, Eγ 77 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 of 197Hg labelled Neohydrin® as radionuclide in nuclear medical diagnostics of the kidney (Greif et al. 1956). The 197Hg labelled Neohydrin® was produced by n/gamma reaction of enriched 196Hg in a reactor, wherein a low specific activity of 1 GB/μmol was achieved. Furthermore, the product was contaminated with 203Hg.
Alternatively, Walther et al. proved the feasibility of the production of the no carrier added (NCA) radionuclide 197mHg 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) radionuclide 197mHg was carried out through proton induced nuclear reactions on gold via the 197Au(p,n)197(m)Hg reaction in quantities up to about each 100 MBq, wherein Au superseded the expensive enrichment for the target material.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 of 197(m)Hg and 197Hg 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 stable 197(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 derivative 203Hg-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 acetate 197Hg (hydroxy(2-hydroxypropyl)197mercury, Merprane®) or Merisoprol acetate 203Hg, respectively, has been used for diagnosis of renal function.
Disadvantages of the disclosed organometallic mercury compounds are the contamination with 203Hg and the toxicity because of the high Hg content.
The invention has the object of finding organometallic 197(m)Hg compounds with high purity and high specific activity.
The objective of the invention is solved by a 197(m)Hg compound according to formula (E)
Ar-197(m)Hg—Y (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 R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -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
For example, the phenyl ring in formula (I) is substituted with 1 to 5 Xn, wherein Xn is selected from X1, X2, X3, X4, X5. Thus, X1 to X5 are independently selected from H, unsubstituted or substituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups.
197(m)Hg according to the invention is a radionuclide comprising at least one of the two radioactive, γ-emitting nuclear isomers 197Hg in the ground state and 197(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 isotope 197Hg 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 radionuclide 197(m)Hg comprises a molar ratio of 197(m)Hg to 197Hg of 1:1 to 2:1.
Advantageously, the contamination of the 197(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 example 194Hg, 195Hg and 203Hg) is less than 10−6% of the 197(m)Hg content (w/w). Preferably the content of non-radioactive Hg isotopes (196Hg, 198Hg, 199Hg, 200Hg, 201Hg, 202Hg and 204Hg) is below the detection limit of inductively coupled plasma mass spectrometry (ICP-MS) of 1·10−12 (w/w).
Preferably the 197(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)2 or —NHR2), nitro (—NO2), phosphate groups or halogenides, wherein R2 is 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 Xn comprises between 1 and 50 carbon atoms, preferred between 1 and 25 carbon atoms, especially preferred between 1 and 10 carbon atoms.
In some embodiments Xn is selected from unsubstituted or substituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups.
In a preferred embodiment Xn or 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 Xn comprises 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 R1 is 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 the 197(m)Hg compound according to formula (I) is substituted with 1 to 3 Xn, wherein Xn is selected from X1, X2 and X3 as described above. In a mostly preferred embodiment the 197(m)Hg compound according to formula (I) is substituted with one Xn or X, respectively, as shown in formula (I′)
wherein X is selected from H, unsubstituted or substituted alkyl, -alkoxy (—OR1), -amide (—CON(R1)2), -carboxy (—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 R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -aryl or -heteroaryl groups.
In a further embodiment the 197(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 X is selected from H, unsubstituted or substituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—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 R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -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 R3 is selected from H, unsubstituted or substituted alkyl, alkoxy (—OW), amide (—CON(R4)2), carboxy (—COOR4), aryl or heteroaryl groups,
wherein R4 is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups. The two R3 are selected independently.
In a further embodiment R3 is selected from unsubstituted or substituted alkyl, alkoxy (—OW), amide (—CON(R4)2), carboxy (—COOR4), aryl or heteroaryl groups.
In a preferred embodiment R3 of 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)
—SR5 (III),
wherein R5 is selected from H, unsubstituted or substituted alkyl, alkoxy (—OR6), amide (—CON(R6)2), carboxy (—COOR6), aryl or heteroaryl groups,
wherein R6 is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups.
In a further embodiment R5 of 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 R7 is selected from H, unsubstituted or substituted alkyl, alkoxy (—OR8), amide (—CON(R8)2), carboxy (—COOR8), aryl or heteroaryl groups,
wherein R8 is 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 R7 of the unsubstituted or substituted phenyl groups is selected from substituted amide (—CON(R8)2) or carboxy (—COOR8) groups.
In a further embodiment the 197(m)Hg compound according to formula (IV) is substituted with R7 in ortho-, meta- or para-position.
In a further embodiment Y is selected from unsubstituted or substituted phenyl groups according to formula (IV), wherein R7 and Xn are 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 R7 and X are identically resulting in a compound according to formula (V)
wherein X is selected from H, unsubstituted or substituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups,
wherein R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -aryl or -heteroaryl groups.
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 R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -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.
In some embodiments the resulting 197(m)Hg-compounds have one of the following formulas:
Preferably, in the 197(m)Hg compound according to the invention, both 197(m)Hg-substituents are linked by at least one aliphatic and/or aromatic spacer molecule as shown in Formula Ebridge:
“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.
“Both 197(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: CH2 at each end of the spacer).
In embodiments the spacer molecule comprises a reactive group, for example, OH, SH, NH2 or 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 R2 is H or alkyl, and
R3 is 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, NH2 and COOH,
and the targeting moiety being selected from a nucleic acid, antibody, antibody fragment, peptide, and oligonucleotide; and
R4 and R4′ are independently selected from H and Aryl.
Alkyl can be n-butyl especially, or other groups resulting from coupling with Li-organyls.
For example, in some embodiments, in formula (VIIIBridge) R3 is H, R2 is n-Butyl and both R4 and R4′ are Phenyl.
Preferably, in the 197(m)Hg compound according to the invention, both 197(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 both 197(m)Hg-substituents in ortho position to the bond to 197(m)Hg.
The 197(m)Hg compound according to the invention is more preferably a compound of formula (VII)
Alkyl can be n-butyl especially, or other groups resulting from coupling with Li-organyls.
In further embodiments of the invention both 197(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 R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -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 the 197(m)Hg-moiety, at the aryl or heteroaryl groups of formulas (Ibridge), (Iabridge), (Ibbridge) or (Icbridge).
In a preferred embodiment the phenyl groups of the 197(m)Hg compound according to the invention are linked by at least one aliphatic and/or aromatic spacer molecule as shown in formula (VI)
wherein X is selected from H, unsubstituted or substituted alkyl, alkoxy (—OR1), amide (—CON(R1)2), carboxy (—COOR1), aryl or heteroaryl groups,
wherein R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -aryl or -heteroaryl groups.
In an embodiment the 197(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 Xn and/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 a further embodiment Xn and/or Y comprise at least one amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue or aliphatic spacer, preferably Xn and/or Y comprise 1 to 3 amino acids, peptides, proteins, antibodies, oligonucleotides, alkaloid residues or aliphatic spacers. In a preferred embodiment Xn or Y comprises one amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue or aliphatic spacer.
In an embodiment Xn and/or Y comprises one aliphatic spacer and one amino acid, peptide, protein, antibody, oligonucleotide or alkaloid residue.
Advantageously, the 197(m)Hg compounds according to the invention exhibit high purity and high specific activity.
As used herein, the term “purity” refers to the amount of 197(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 the 197(m)Hg compound according to the invention is based on the molar amount of the 197(m)Hg compound. The specific activity can be determined for example by inductively coupled plasma mass spectrometry (ICP-MS).
In an embodiment the 197(m)Hg compounds according to the invention have a specific activity of at least 100 GBq/μmol based on the molar amount of the 197(m)Hg compound, i.e. of mercury, preferred 100 to 1.000 GBq/μmol based on the molar amount of the 197(m)Hg compound.
In an embodiment the 197(m)Hg compounds of the invention can be used in nuclear medical diagnostics and endoradionuclide therapy (theranostic).
In a further embodiment the 197(m)Hg compounds of the invention can be used in the treatment of cancer.
In a further embodiment the 197(m)Hg compounds of the invention can be used for the manufacture of a medicament for endoradionuclide therapy.
In a further embodiment the 197(m)Hg compounds of the invention can be used for the manufacture of a medicament for the treatment of cancer.
In a further embodiment the 197(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 a 197(m)Hg compound of the invention.
The present invention further comprises a method for the production of the 197(m)Hg compounds according to the invention comprising the steps:
Advantageously, the method for the production of 197(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 isotopes 197Hg and 197(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 of 197(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 of 197(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 R1 is 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,
R10 is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups, preferred C1 to C5-alkyl groups;
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,
wherein R1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -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,
R10 is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups, preferred C1 to C5-alkyl groups;
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):
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):
In a further embodiment the synthesis of NCA 197(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 NCA 197(m)Hg synthesised according to step b) is NCA 197(m)HgCl2.
In a further embodiment the synthesis of NCA 197(m)Hg according to step b) is followed by purification of the NCA 197(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 NCA 197(m)HgCl2 to 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 NCA 197(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 symmetric 197(m)Hg compounds.
As used herein, the term “symmetric 197(m)Hg compounds” refers to 197(m)Hg compounds, wherein 197(m)Hg exhibits two identical binding partners. Symmetric 197(m)Hg compounds are 197(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 asymmetric 197(m)Hg compounds.
As used herein, the term “asymmetric 197(m)Hg compounds” refers to 197(m)Hg compounds, wherein 197(m)Hg exhibits two different binding partners. Asymmetric 197(m)Hg compounds are 197(m)Hg compounds of the invention, except 197(m)Hg compounds according to formula (V) and (VI).
In a further embodiment the formed asymmetric 197(m)Hg compounds are added to dithiocarbamate ligands to form 197(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.
As used herein, the term “activated ester groups” refers to N-hydroxysuccinimide (NHS) or tetrafluorophenyl (TFP) ester groups.
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 the 197(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 the 197(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 of 197(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 the 197(m)Hg compounds according to the invention.
A pharmaceutical composition containing the 197(m)Hg compounds according to the invention typically contains a pharmaceutically acceptable carrier, such as saline. The dose of the 197(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 the 197(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 the 197(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 the 197(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 the 197(m)Hg compounds according to the invention will be about 5 to 50 MBq/kg.
The pharmaceutical compositions and formulations containing the 197(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 the 197(m)Hg compounds according to the invention are prepared for administration and/or storage by mixing the 197(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). The 197(m)Hg compounds according to the invention can be administered alone, or in various combinations, and in combination with other therapeutic agents. The 197(m)Hg compounds used in the invention are normally stored in solution.
Preferably the 197(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 the 197(m)Hg-compound and for the corresponding organic precursor compound as well.
The present invention will now be further explained by the following non-limiting figures and examples.
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 decaying 197(m)Hg. ICP-MS measurements were carried out on an ELAN 9000 (PerkinElmer SCIEX, Waltham, USA).
Gamma-Ray Spectroscopy
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 and 13C 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 the 197(m)Hg compound divided by the specific activity of the no carrier added (NCA)197(m)Hg.
High-Performance Liquid Chromatography (HPLC) Measurements
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%.
Mass Spectrometry (Electrospray Ionization (ESI)-MS, Matrix-Assisted Laser Desorption/Ionization (MALDI)-MS)
For mass spectrometry a QuadroLC by Micromass with electrospray ionisation (ESI) mode and a Bruker MALDI-TOF MS instrument (MALDI) were used.
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).
1H NMR (400 MHz, CDCl3) δ (ppm): 8.23 (s, 1H), 7.92 (dd, J=7.8, 1.6 Hz, 2H), 7.64 (s, 2H), 7.59 (d, J=7.9 Hz, 2H), 7.48 (t, J=7.8 Hz, 1H), 7.28 (s, 1H), 7.04 (d, J=7.8 Hz, 2H), 6.84 (d, J=5.3 Hz, 2H), 4.52 (d, J=5.8 Hz, 4H),
13C NMR (101 MHz, CDCl3) δ (ppm): 166.57, 140.37, 136.93, 134.52, 130.64, 130.40, 129.27, 127.32, 125.67, 94.78, 43.61.
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).
1H NMR (400 MHz, d6-DMSO) δ (ppm): 9.11 (broad t, 2H), 8.38 (s, 1H), 8.01 (dd, J=7.8, 1.6 Hz, 2H), 7.58 (t, J=7.8 Hz, 1H), 7.53-7.22 (m, 8H), 4.47 (d, J=5.9 Hz, 4H), 0.25 (s, 18H),
13C NMR (101 MHz, CDCl3) δ (ppm): 166.41, 143.39, 137.31, 135.72, 135.46, 134.92, 130.12, 129.18, 128.61, 128.22, 125.51, 44.68, −9.35.
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 of 197(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.
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.
C22H16HgN2O8, Chemical Formula:
Molecular Weight: 636.97 g/mol,
1H-NMR (400 MHz, DMSO-D6) δ (ppm): 2.89 (s, 8H); 7.77 (d, 4H); 7.99 (d, 4H),
13C-NMR (100 MHz, DMSO-D6) δ (ppm): 25.5 (CH2); 123.5 (C); 128.8 (CH); 137.8 (CH); 161.9 (C); 162.0 (C); 170.3 (C), yield: 7 mg (15.4 μmol; 77%),
ESI+ m/z: 637 [M]+; 539 [M-NHS]+.
The 197(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 buffered 197(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).
The 197(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 buffered 197(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).
Radiochemical yield (TLC): ≥95%,
Radiochemical purity (TLC): ≥95%
Radio-TLC: Rf=0.45 (ACN/H2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
(See Ref. Partyka et al., J. Organometallic Chemistry):
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.
C8H6HgS2, Chemical Formula:
Molecular Weight: 366.85 g/mol,
ESI+ m/z: 369 [M]+.
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.
C10H6HgO4S2, Chemical Formula:
Molecular Weight: 454.86 g/mol,
ESI+ m/z: 457 [M]+.
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.
C20H18Fe2Hg, Chemical Formula:
Molecular Weight: 570.64 g/mol,
ESI+ m/z: 573 [M]+.
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.
C12H8HgN2O4, Chemical Formula:
Molecular Weight: 444.02 g/mol,
ESI+ m/z: 447 [M]+.
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.
C11H8HgO2S, Chemical Formula:
Molecular Weight: 404.83 g/mol,
ESI+ m/z: 407 [M]+.
Based on B-Precursors
A solution of 10-100 μg aryl boronic acid precursor in 50-100 μl ethanol is added to the intended amount 197(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).
Radiochemical yield (TLC): ≥95%,
Radio-TLC: Rf=0.2 (ACN/H2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
Radiochemical yield (TLC): ≥95%,
Radio-TLC: Rf=0.9 (ACN/H2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
Radiochemical yield (TLC): ≥95%,
Radio-TLC: Rf=0.1 (ACN/H2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
Radiochemical yield (TLC): ≥95%,
Radio-TLC: Rf=0.9 (ACN/H2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
This heteroleptic diaryl mercury compound is accessible in a two-step procedure (analogous to the asymmetric phenylmercury dithiocarbamate derivatives (see next section):
The 197(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 acidic 197(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).
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 the 197(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).
Radiochemical yield (TLC): ≥60%,
Radio-TLC: Rf=0.45 (ACN/H2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
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 (see
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.
C14H10HgO4, Chemical Formula:
Molecular Weight: 442.82 g/mol,
1H-NMR (400 MHz, DMSO-D6, AcOH-D4) δ (ppm): 7.52 (d, 4H); 7.83 (d, 4H),
13C-NMR (100 MHz, DMSO-D6, AcOH-D4) δ (ppm): 129.6 (CH); 131.0 (C); 137.7 (CH); 161.6 (C); 168.4 (C), yield: 15.3 mg (34 μmol; 94%),
ESI+ m/z: 443 [Hg-M]+.
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.
Radiochemical yield (TLC): ≥95%,
Radiochemical purity (TLC): ≥95%
Radio-TLC: Rf=0.6 (ACN/H2O 90:10 (v/v) with 0.1 vol-% TFA, RP-18).
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.
Radiochemical yield (TLC): ≥50-70%,
Radiochemical purity (TLC): ≥95%,
Radio-TLC: Rf=0 (ACN/H2O 90:10 (v/v) with 0.1 vol-% TFA, RP-18).
The synthesis is schematically shown in the following scheme:
Compound (3*) was characterized by UV (
In vivo stability of (3*) was tested (
The results (
To test the actual in vivo stability of 3*, a biodistribution was performed on healthy rats and the results (
A 250 ml round-bottomed flask, with magnetic flea, was charged with 1,3-diphenylpropan-2-one (8.03 g, 38.2 mmol), followed by THF (100 ml) and stirred until a clear, pale yellow solution formed. To this was added 2-bromobenzylamine (14.20 g, 76.3 mmol, Alfa Aesar), a 37 wt % aqueous solution of formaldehyde (11.4 ml, 152.6 mmol) and a catalytic amount of ethanoic acid (a few drops). The reaction mixture was refluxed at 65° C. overnight for 19 h forming a dark yellow solution. TLC confirmed the complete conversion of the ketone starting material (Rf≈0.8, 1:1 EtOAc:hexane, KMnO4 stain). The ethanoic acid was neutralized by adding saturated Na—HCO3(aq) until the reaction mixture was slightly alkaline. The THF was removed by evaporation, the reaction mixture dissolved in DCM (50 ml) and washed with water (3×20 ml). The aqueous layers were combined and extracted with DCM (5 ml). The organic layers were combined, washed with brine (10 ml), dried with anhydrous Na2SO4 and filtered. The DCM was evaporated, leaving a brown solid, which was recrystallized by dissolving in boiling EtOH and slowly cooling to room temperature, affording 1 as white crystals (17.95 g, 28.5 mmol, 75%). TLC Rf≈0.8 (1:1 EtOAc:hexane, KMnO4 stain). >99% HPLC purity. Anal. ESI-MS: calculated [M+H]+ 631.0783, found 631.0781. 1H NMR (400 MHz, CDCl3): δ 7.64 (dd, J=8.0, 1.2 Hz, 2H, Ar), 7.53 (dd, J=7.6, 1.7 Hz, 2H, Ar), 7.34 (td, J=7.5, 1.3 Hz, 2H, Ar), 7.31-7.27 (m, 3H, Ar) 7.24-7.16 (m, 9H, Ar), 3.85 (s, 4H, NCH2Ar), 3.42 (dd, J=186.0, 10.7 Hz, 8H, CCH2N). 13C NMR (101 MHz, CDCl3): δ 210.89 (C═O), 142.75, 137.37, 133.31, 131.71, 129.21, 127.98, 127.41, 127.03, 126.72, 125.21, 64.87 (CCH2N), 61.25 (NCH2Ar), 54.64 (PhCCO).
1 (1.70 g, 2.7 mmol) was charged into an oven-dried 250 ml round-bottomed Schlenk flask with a magnetic flea, on a Schlenk line, under argon and sealed with a rubber septum. Anhydrous THF (50 ml) was syringed into the flask to form a suspension. Carefully, dropwise addition of nBuLi (2.5 M in hexane, 5.4 ml, 13.5 mmol), keeping the temperature below the boiling point, then reacted with the suspension to form a clear yellow solution. Me3SnCl (1 M in THF, 13.5 ml, 13.5 mmol) was syringed drop-wise 30 min later, eventually causing the reaction mixture to turn colorless. After leaving to stir throughout the night (19 h), the reaction mixture was carefully quenched with EtOH and then water. Organic solvents were removed by evaporation. More water was then added, into a final 40 ml solution, then NaHCO3 to form an alkaline phase pH≈8 that was extracted with DCM (3×40 ml). Afterwards, all organic phases were combined and washed with brine solution (40 ml), dried with anhydrous Na2SO4, solids filtered off and the remaining solution evaporated leaving a crude brown oily residue. Recrystallization with Et2O formed a brown ppt. that was filtered off. Evaporation of the remaining Et2O left 2 g of a crude brown oily residue. Column chromatography purification (dry-loaded onto Alox (basic 90) as the desired product proved unstable on silica) with a slow gradient of EtOAc (0% to 5%) in hexane yielded 2 as a white solid (200 mg, 0.23 mmol, 9%). TLC: Rf≈0.7 (Alox plate, 15% EtOAc:hexane, 12 stain). >90% HPLC purity. Anal. ESI-MS: calculated [M+H]+ 857.2666, found 857.2677. 1H NMR (400 MHz, CDCl3) δ 7.80 (t, J=7.0 Hz, 2H, SnAr-o), 7.49-7.11 (m, 16H, Ar), 3.79 (s, 2H, NCH2Ar), 3.77 (s, 2H, NCH2Ar), 3.37 (dd, J=42.7, 11.3 Hz, 4H, CCH2N), 2.88 (dd, J=51.6, 10.6 Hz, 4H, CCH2N), 2.27 (s, 1H, OH), 1.26 (m, 2H, Bu-C1), 0.64 (td, J=14.2, 6.7 Hz, 2H, Bu-C3), 0.44 (s, 9H, SnMe3), 0.38 (s, 9H, SnMe3), 0.36 (t, J=7.3 Hz, 3H, Bu-C4), 0.02 (m, 2H, Bu-C2). 13C NMR (101 MHz, CDCl3): δ 145.29, 145.25, 144.47, 136.22, 136.08, 129.26, 129.02, 128.93, 128.61, 127.85, 127.35, 127.16, 126.84, 126.18, 125.67, 65.61, 64.38, 64.29, 60.30, 47.32, 30.48, 30.24, 29.58, 26.12, 23.34, 13.71, −7.44 (SnMe3), −7.47 (SnMe3).
A 1.5 ml Eppendorf LoBind hinge-top tube was charged with 2 (26 mg, 0.03 mmol) and HgCl2 (8.1 mg, 0.03 mmol) in THF (1.5 ml) and mixed at 50° C. for 5 h. Evaporation of THF left a yellow oily residue. HPLC analysis showed no remaining starting material. Purification by HPLC yielded a white solid (7.2 mg, 5.25 mmol, 33%). ˜90% HPLC purity. Anal. ESI-MS: calculated [M+H]+ 731.2925, found 731.2926. 1H NMR (600 MHz, CDCl3): δ 7.54 (d, J=7.8 Hz, 4H), 7.48 (dd, J=6.9, 1.4 Hz, 1H), 7.45 (dd, J=7.1, 1.4 Hz, 1H), 7.43-7.12 (m, 11H), 7.09 (td, J=7.4, 1.5 Hz, 1H), 3.62 (s, 2H, NCH2Ar), 3.51 (s, 2H, NCH2Ar), 3.33 (dd, J=198.0, 11.5 Hz, 4H, CCH2N), 2.99 (dd, J=305.4, 11.4 Hz, 4H, CCH2N), 1.73 (s, 1H, OH), 1.44 (t, J=8.5 Hz, 2H, Bu-C1), 0.74 (h, J=7.3 Hz, 2H, Bu-C3), 0.40 (t, J=7.3 Hz, 3H, Bu-C4), −0.04 (p, J=7.9 Hz, 2H, Bu-C2). 13C NMR (101 MHz, CDCl3): δ 171.33 (HgC1), 170.92 (HgC1), 146.84 (HgC2), 146.44 (HgC2), 141.78 (Ph-i), 139.08 (HgC6), 138.84 (HgC6), 128.96 (Ph-m), 128.95 (Ph-p), 127.98 (HgC3), 127.83 (HgC3), 127.23 (HgC4), 127.16 (HgC4), 126.72 (HgC5), 126.67 (HgC5), 126.41 (Ph-o), 75.09 (COH), 67.71 (NCH2Ar), 66.99 (NCH2Ar), 60.17 (CCH2N), 60.09 (CCH2N), 46.25 (CPh), 31.39 (Bu-C1), 25.76 (Bu-C2), 25.99 (Bu-C3), 13.62 (Bu-C4). 199Hg NMR (108 MHz, CDCl3): δ −684.5.
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).
| Number | Date | Country | Kind |
|---|---|---|---|
| 17155213.6 | Feb 2017 | EP | regional |
This application is a continuation-in-part of U.S. application Ser. No. 16/478,687 filed Jul. 17, 2019 and published as US 2019/0367537 on Dec. 5, 2019, which is a national stage filing under section 371 of International Application No. PCT/EP2018/052996, filed on Feb. 7, 2018, and published on Aug. 16, 2018 as WO 2018/146116, which claims priority to European Application No. 17155213.6, filed on Feb. 8, 2017. The entire contents of WO 2018/146116 and US 2019/0367537 are hereby incorporated herein by reference. 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 197(m)Hg by electrophilic substitution; and the use of the 197(m)Hg compounds for nuclear medical diagnostics and endoradionuclide therapy (theranostic), particularly the treatment of cancer.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16478687 | Jul 2019 | US |
| Child | 17159968 | US |
