DE102009029033A1 - Tetrathiol ligands, metal complexes, conjugates and kits, their use in nuclear medicine diagnostics and endoradione nuclide therapy, as well as methods of making tetrathiol ligands and metal complexes - Google Patents

Abstract

A ligand of formula $ F1 wherein (CH-R1) and (CH-R1 ') represent substituted or unsubstituted methylene bridges, each of R1 and R1', and R3 and R3 'are each independently selected from hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups, m and n are positive integers, where m represents the number of groups (CH-R1 ') and n represents the number of groups (CH-R1), the sum of m and n ranging from 4 to 10 R2 and R2 'are independently selected from hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted carboxyl groups, substituted or unsubstituted aryl groups or Z, X is a substituted or unsubstituted methylene bridge of the formula CHR4 or an unsubstituted amino group (NH) or substituted amino group (NZ) wherein Z is - (CH-R5) pA, wherein (CH-R5) is a substituted or unsubstituted methylene and p is an integer representing the number of groups (CH-R5), where p ranges from 2 to 8, A represents a coupling unit, R4 and the individual R5 are each independently selected Hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups.

Description

The The invention relates to the field of radiopharmacy, in particular metal complexes of tetrathiol ligands and their use as radioactive drugs for diagnostics and in endoradione nuclide therapy as well a process for the preparation of tetrathiol ligands and metal complexes.

Diagnosis with technetium-99m preparations is widely used to study the renal and hepatobiliary system, skeleton, myocardium and brain. Rhenium isotopes are of interest for therapeutic use in endoradione nuclide therapy. The β-emitting isotope ¹⁸⁸ Re (E _max = 2.12 MeV) is an attractive radioisotope for radiotherapy and radioimmunotherapy, as it is slightly carrier-less than perrhenation (ReO ₄ ^- ) in aqueous solution from a ¹⁸⁸ W / ¹⁸⁸ Re generator system, except for its radiation-relevant radiative properties can be obtained Guhlke S, Beets AL, Oetjen K, Mirzadeh S, Biersack HJ, Knapp FF Jr. Simple new method for effective concentration of 188Re solutions from alumina-based 188W-188Re generator. J Nucl Med. 2000, 41 (7): 1271-8. ]. Due to the generator's long lifetime, ¹⁸⁸ Re is available at a low cost for the routine preparation of radiopharmaceuticals for the treatment of, for example, tumors.

Regardless, there are only a limited number of rhenium supplements such as ¹⁸⁶ Re-DMSA, ¹⁸⁶ Re-DTPA, and ¹⁸⁶ re-diphosphonates for endoradione nuclide therapy. However, these compounds are insufficient for therapeutic use because they are too non-specific.

One Another limiting factor in the development of Rheniumradiophamaka is the lack of suitable complexing agents with which the radioactive Metal stable to other molecules, especially biomolecules, can be coupled. In particular, the stability many rhenium chelates in terms of hydrolysis, re-oxidation to perrhenate and radiolysis resulting in the release of radioactive metal from the targetbindenden radiopharmaceutical runs.

Derivatives of dimercaptosuccinic acid have been proposed as agents for the labeling of proteins and antibodies with technetium-99m and rhenium-186 and rhenium-188, respectively ( US Pat. No. 5,175,256 , 1992). Even this technical solution does not lead to clearly defined connections.

Regarding The above stability criteria are satisfied by the described as well as other systems yet the requirements for a wide in vivo application.

Recently, the inventors described tetrathiol ligands that are accessible by bridging two DMSA molecules ( WO 05108330 A1 ). The corresponding technetium and rhenium complexes show a very high chemical and radiolytic stability. However, a disadvantage of this ligand system is the complicated organic synthesis over several stages and the resulting low yields.

task The invention is to provide improved tetrathiol ligands and their radiolytic and metabolically stable metal complexes used for conjugation are suitable with biomolecules, as well as methods for producing the Specify chelating ligands and metal complexes.

Solved the object is achieved by the ligands described in claim 1, as well as their use for binding of radiometals, preferably Technetium and rhenium, to biomolecules.

worin (CH-R1) und (CH-R1') substituierte oder unsubstituierte Methylenbrücken darstellen.The invention relates to a ligand of the formula

wherein (CH-R1) and (CH-R1 ') represent substituted or unsubstituted methylene bridges.

The individual R1 and R1 ', and R3 and R3' are each independently selected Hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups. Preferred R 1 and R 1 'as well as R 3 and R 3' are each hydrogen and C 1 to C 3 alkyl, more preferably methyl and ethyl.

m and n are positive integers, where m is the number of groups (CH-R1 ') and n represents the number of groups (CH-R1). The sum of m and n is preferably in the range of 4 to 10, more preferably n and m are each selected from 2, 3 and 4.

R2 and R2 'are independently selected from hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted carboxyl groups, substituted or unsubstituted aryl groups or those defined below Group Z. Preferred R2 and R2 'are independent of each other selected from -COOH, ester groups and substituted or unsubstituted acid amide groups.

X is a substituted or unsubstituted methylene bridge with the formula CHR4 or an unsubstituted amino group (N-H) or substituted amino group (N-Z).

Z is - (CH-R5) p-A, wherein (CH-R5) is a substituted one or unsubstituted methylene bridge and p is a integer is the number of groups (CH-R5) stands. Preferably, p is in the range of 2 to 8. Particularly preferred p is selected from 4, 5 or 6.

A represents a pairable unit, i. H. a functional Group, which is the simple attachment of an organic molecule or biomolecules allowed. A is preferably selected from ester groups, acid amide groups, isothiocyanate groups (-NCS), Thiol groups, amino groups, hydroxyl groups and halogens, preferably Bromine or iodine.

R4 and the individual R5 are each independent of each other are selected from hydrogen, substituted or unsubstituted Alkyl groups, substituted or unsubstituted aryl groups. Preferred R4 and R5 are each hydrogen and C1 to C3-alkyl, particularly preferably methyl and ethyl.

The Ester groups in R2, R2 'and A are preferably carboxylic acid ester groups of the COOR type with R = alkyl or aryl, preferably C1 to C7 alkyl or aryl, particularly preferably selected from methyl, ethyl, n-propyl, Isopropyl, n-butyl, tert-butyl, phenyl, benzyl, tosyl, alkylsulfonyl, Polyethylene glycol (PEG).

The acid amide groups in R ₂ , R ₂ 'and A are preferably carboxylic acid amide groups of the type CON (R) ₂ where R = hydrogen, alkyl, alkoxy or aryl, preferably C 1 to C 7 alkyl or aryl, more preferably independently selected from methyl, ethyl, n Propyl, isopropyl, n -butyl, tert -butyl, phenyl, benzyl, tosyl, alkylsulphonyl, PEG.

Alternatively, the two R in the acid amide group CON (R) ₂ together with the N form an acyclic or cyclic radical having preferably 3 to 6 C atoms and optionally 1 to 6 further heteroatoms (preferably selected from N, S and O). Particularly preferred acyclic and cyclic radicals are selected from:

One Another object of the invention is a conjugate of an inventive Ligands and an organic molecule or biomolecule, wherein the attachment of the organic molecule or biomolecule via the functional group A takes place in X, R2 or R2 '.

Prefers is the organic molecule or biomolecule in the Conjugate selected from proteins, sugars, lipids, nucleic acids, Antibodies, peptides and oligonucleotides, as well as small ones organic molecules, in particular cytostatics. The tailed Organic molecules or biomolecules are preferably target-seeking Molecules (targeting units), d. H. molecules advantageous to a particular target, d. H. certain Cells or organs, in the body selectively bind by the Connection of these molecules will make it possible radioactive complex targeted to specific organs or cells, z. As carcinomas, by the connection of cytotoxic agents such. B. (Tamoxifen) to conduct.

Of the Term antibody in the sense of the present invention includes besides monoclonal and polyclonal antibodies also recombinant antibodies and fragments, such as. ScFv (single-chain fragments) and Fab fragments. The term nucleic acids within the meaning of the invention, besides Desoyribonukleinsäuren (DNA) and ribonucleic acids (RNA) also all other linear Polymers in which the bases adenine (A), cytosine (C), guanine (G) and thymine (T) or uracil (U) arranged in a corresponding sequence are, in particular modified nucleic acids Backbone, such as. B. a phosphothioate, phosphoramidate or O-methyl derivatized backbone, peptide nucleic acids (PNA), and locked nucleic acids (LNA).

worin (CH-R1) und (CH-R1') einfach substituierte oder unsubstituierte Methylenbrücken darstellen, die einzelnen R1 und R1', R2 und R2', R3 und R3', m und n, sowie X jeweils unabhängig voneinander und wie oben definiert ausgewählt sind.The invention also includes metal complexes of the formula

wherein (CH-R1) and (CH-R1 ') represent monosubstituted or unsubstituted methylene bridges, the individual R1 and R1', R2 and R2 ', R3 and R3', m and n, and X are each independently and as defined above are selected.

Y is a metal atom or a metal complex, preferably an oxo or nitrido complex of a metal. Particularly preferably, Y is selected from ^99m TcO, ¹⁸⁶ ReO, ¹⁸⁸ ReO, ^99m TcN, ReN ^{186, 188} ReN, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, Sn, Re, and Tc ^{3+ + 3,} preferably Sn (IV).

The Metal complexes with the radionuclides are suitable for the Radiodiagnosis (especially with Tc) and radiotherapy (in particular with Re).

The metal complex according to the invention with tin is particularly suitable for the preparation of a nonradioactive kit, since the tin coordinately binds the four sulfur atoms in the ligand and thereby protects against oxidation. Advantageously, the Sn in the complex can easily be displaced by technetium or rhenium, whereby the corresponding complex with ^99m TcO, ¹⁸⁶ ReO, ¹⁸⁸ ReO, ^99m TcN, ¹⁸⁶ ReN or ¹⁸⁸ ReN arises.

object The invention therefore also relates to the use of tin for stabilization of tetrathioligands, especially in the field of radiopharmacy.

As the ligands according to the invention are also suitable the metal complexes according to the invention for conjugation with a biomolecule. Again, the coupling takes place via the functional group A in X, R2 or R2 '. The biomolecule is preferably selected as described above.

object The invention also relates to the use of an inventive Ligands or conjugates for the preparation of a metal complex.

One Another object of the invention is the use of an inventive Ligands or metal complex for coupling to biomolecules.

An important feature of the compounds of the invention is that they can be prepared via a - compared to the known bridged DMSA ligands - easier synthesis, the corresponding technetium and rhenium, however, have a comparatively high chemical and radiolytic stability. Furthermore, the formation of isomers is specifically controlled by a special embodiment of the complexing agent In contrast to the meso-DMSA, the dimercaptomaleic acid (DMMA) used in the invention is an achiral compound, ie it contains no stereocenters. Therefore, only one isomer is formed when bridging two DMMA units.

By bridging two separate DMMA molecules via a modified Carbon chain in the invention is a new complexing agent synthesized, which enabled four to coordinate to a metal Contains mercapto groups, and this is combined with technetium (V) or rhenium (V) converted to the corresponding complex. The length the carbon chain is chosen so that the mercapto groups in sterically favorable arrangement to the metal stand. In the Carbon chain is a group (X) incorporated. The coupling of Biologically active units take place either via the Group (X) or via R2 or R2 '.

One Another object of the invention is therefore a process for the preparation the ligands according to the invention with the following steps:

• a.) Implementation of
  
  and if necessary (if not R2 = R2 ') implementing
  
  Possible alternatives to thionyl chloride are: PCl ₃ , PCl ₅ , POCl ₃ .
• b.) reacting the acid chloride obtained in step a.) with R3- (NH) - (CH-R1) _n -X- (CH-R1 ') _m - (NH) -R3'
  
• c.) Sulfur substitution by oxygen too
  
  preferably in the presence of a mercury salt, particularly preferably Hg (II) acetate;
• d.) hydrolysis to
  
  preferably in the presence of an alkali metal hydroxide, particularly preferably NaOH or KOH.

(CH-R1) and (CH-R1 ') each represent monosubstituted or unsubstituted ones Methylene bridges. The individual R1 and R1 ', R2 and R2', R3 and R3 ', m and n, and X are each independently and selected as defined above.

step b.) is preferably carried out under protective gas and preferred at temperatures from -20 ° C to 10 ° C.

bevorzugt mit R2 und R2' unabhängig voneinander ausgewählt aus -COOH, Estergruppen und substituierten oder unsubstituierten Säureamidgruppen, geschieht bevorzugt durch:

• a.) Umsetzen von Dialkylacetylendicarboxylat mit Ethylentrithiocarbonat zum 2-Thioxo-1,3-dithiol-4,5-dicarbonsäuredialkylester, wobei Alkyl bevorzugt ausgewählt ist aus C1-C3, besonders bevorzugt Methyl,
• b.) Hydrolyse des Diesters zur Dicarbonsäure, bevorzugt durch Zugabe einer Säure,
• c.) Kondensation der Dicarbonsäure zum Anhydrid, bevorzugt durch Zugabe von Essigsäureanhydrid (vorzugsweise unter Schutzgas)
• d.) Umsetzen des so erhaltenen 2-Thioxo-1,3-dithiol-4,5-dicarbonsäure-Anhydrids mit
• e.) einem Alkohol HOR bzw. Amin zum Carbonsäurester des Typs COOR oder Carbonsäureamiddes Typs CON(R)₂, wobei R ausgewählt ist, wie zu R2, R2, R2' und A weiter oben angegeben.

The preparation of the starting compounds used in the process according to the invention

preferably with R2 and R2 'independently of one another selected from -COOH, ester groups and substituted or unsubstituted acid amide groups, is preferably carried out by:

• a.) reacting dialkylacetylenedicarboxylate with ethylenetrithiocarbonate to give the 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid dialkyl ester, where alkyl is preferably selected from C 1 -C 3, particularly preferably methyl,
• b.) hydrolysis of the diester to the dicarboxylic acid, preferably by addition of an acid,
• c.) condensation of the dicarboxylic acid to the anhydride, preferably by addition of acetic anhydride (preferably under protective gas)
• d.) reacting the thus obtained 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid anhydride with
• e.) an alcohol HOR or amine to the carboxylic acid ester of the type COOR or carboxylic acid amide of the type CON (R) ₂ , wherein R is selected as indicated to R2, R2, R2 'and A above.

are R2 and R2 'free carboxyl groups (-COOH) omitted the steps c.) and d.).

1 shows a simplified schematic overview of the preparation process according to the invention using, for example, R1 = R1 '= R3 = R3' = H, n = m = 3 and R2 = R2 '= C = OR, where R is the abovementioned OR (from COOR) or N ( R) ₂ (from CON (R) ₂ ), as well as the preparation of the complex according to the invention with Sn or Oxotechnetium (M = Tc) or Oxorhenium (M = Re).

• a.) eines erfindungsgemäßen Liganden oder dessen Konjugat mit einem Biomolekül, eines Komplexes mit Y ausgewählt aus Sn und/oder ein Konjugats mit einem Zinnkomplex, mit
• b.) einem Metallsalz, bevorzugt Pertechnetat oder Perrhenat, in Gegenwart eines Reduktionsmittels, bevorzugt einem Zinn(II)salz (wie z. B. SnCl₂).

The invention also relates to a method for producing a radioactively labeled metal complex or conjugate by reacting

• a.) Of a ligand according to the invention or its conjugate with a biomolecule, a complex with Y selected from Sn and / or a conjugate with a tin complex, with
• b.) a metal salt, preferably pertechnetate or perrhenate, in the presence of a reducing agent, preferably a tin (II) salt (such as SnCl ₂ ).

For the preparation of the nitrido complexes (preferably ^99m TcN, ¹⁸⁶ ReN or ¹⁸⁸ ReN) a nitrogen source, preferably dialkyldithiocarbazate, is added to the reaction mixture in addition to components a.) And b.).

Prefers are contained in the reaction solution to stabilizers, that stabilize the tin salt, z. B. Tatrat, and possibly also stabilizers for the rhenium, z. As ascorbic acid.

The Preparation of radioactively labeled metal complex or conjugate with technetium can be done advantageously at room temperature and is preferably carried out at 15 to 40 ° C.

For the preparation of the conjugates of metal complex and biomolecule according to the invention preferably the non-radioactive ligand or the corresponding tin complex is coupled to the biomolecule and then the radioaktiev metal or metal complex (preferably ^99m TcO, ¹⁸⁶ ReO, ¹⁸⁸ ReO, ^99m TcN, ¹⁸⁶ ReN or ¹⁸⁸ ReN) bound to the ligand. To prepare the conjugate according to the invention from a tin complex and a biomolecule, the tin complex is preferably first prepared and the complex is bound to the biomolecule via the functional group A in X, R2 or R2 '.

• a.) einen erfindungsgemäßen Liganden und/oder einen erfindungsgemäßen Metallkomplex mit Zinn (bevorzugt Sn(IV)),
• b.) Kopplungsreagenzien (wie z. B. BOP, HBTU, HATU, DCC)
• c.) Puffersubstanzen,
• d.) gegebenenfalls Stabilisatoren und Hilfsstoffe wie Tartrat, Glucose, Mannose, Mannitol, Ascorbinsäure.

The invention also provides a kit for producing a conjugate containing

• a) a ligand according to the invention and / or a metal complex according to the invention with tin (preferably Sn (IV)),
• b.) Coupling reagents (such as BOP, HBTU, HATU, DCC)
• c.) buffer substances,
• d) optionally stabilizers and excipients such as tartrate, glucose, mannose, mannitol, ascorbic acid.

• a.) einen erfindungsgemäßen Liganden, dessen Konjugate mit einem Biomolekül und/oder einen erfindungsgemäßen Metallkomplex mit Zinn (bevorzugt Sn(IV)) oder dessen Konjugate,
• b.) Reduktionsmittel, bevorzugt ausgewählt aus Zinn(II)salzen (wie z. B. SnCl₂),
• c.) gegebenenfalls eine Stickstoffquelle
• d.) Puffersubstanzen,
• e.) gegebenenfalls Stabilisierungsreagenzien, bevorzugt auswählt aus Tartrat, Oxalsäure und Ascorbinsäure
• f.) gegebenenfalls Hilfstolle wie Glucose, Mannose, Mannitol, gamma-Cyclodextrin.

Another object of the invention is a kit for preparing a metal complex or its conjugates containing

• a.) a ligand according to the invention, the conjugates thereof with a biomolecule and / or a metal complex according to the invention with tin (preferably Sn (IV)) or its conjugates,
• b.) reducing agents, preferably selected from tin (II) salts (such as, for example, SnCl ₂ ),
• c.) optionally a nitrogen source
• d.) buffer substances,
• e.) optionally stabilizing reagents, preferably selected from tartrate, oxalic acid and ascorbic acid
• f.) optionally auxiliary roles such as glucose, mannose, mannitol, gamma-cyclodextrin.

The The invention also encompasses the use of a device according to the invention Ligands, a metal complex of the invention or their conjugates or an inventive Kits in nuclear medicine, especially in nuclear medicine Diagnostics and in endoradione nuclide therapy.

EXAMPLES

The Invention will be explained below by way of examples without being limited to these.

The process for the preparation of the ligands according to the invention and their technetium and rhenium complexes is explained below by the specific example - s. Reaction scheme in 2 , Preparation of 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid dimethylester 1

10 g (70 mmol) of dimethyl acetylenedicarboxylate and 9.59 g (70 mmol) of ethylenedithiocarbonate are dissolved in 35 ml of toluene in each case. Subsequently, both solutions are combined and heated at 120 ° C for 7 h under reflux condition. During the reaction, a color change from yellow to orange takes place. After heating, the reaction mixture is stirred for a further 12 h at RT. Subsequently, the reaction mixture is concentrated in a rotary evaporator to half and allowed to crystallize at 4 ° C to crystallize. Successive concentration of the filtrate gives further product. By combining the first two solids is obtained 11.07 g of compound 1 as a yellow, finely crystalline solid. Preparation of 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid monohydrate 2

4.16 g (16.6 mmol) of dimethyl ester 1 are suspended in a mixture of 64 ml of concentrated hydrochloric acid and 64 ml of concentrated acetic acid. The reaction mixture is stirred for 4 days at 40.degree. The reaction mixture is then cooled slowly to room temperature to give an orange-yellow, fine precipitate. At 4 ° C, the further crystallization of the product takes place. The recovered solid is filtered off and the resulting filtrate concentrated by 50% on a rotary evaporator. Due to the successive concentration further product is obtained. 3.66 g of compound 2 are obtained as an orange-yellow solid. Preparation of 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid anhydride 3

1.06 g (4.41 mmol) of dicarboxylic acid 2 are stirred in 6.24 ml (66 mmol) of acetic anhydride as reactant and solvent under a protective gas atmosphere for 1.5 hours at a temperature of 40 ° C. Subsequently, the reaction mixture is allowed to cool to room temperature, whereby a fine crystalline yellow solid precipitates. Concentration of the solution to dryness on a rotary evaporator (argon aeration) gives 961.15 mg of a yellow finely crystalline solid. Preparation of 5- (morpholine-N-carbonyl) -2-thioxo-1,3-dithiol-4-carboxylic acid 4

There are 905 mg (4.43 mmol) of the anhydride 3 dissolved in 30 ml of anhydrous dichloromethane and placed in the flask. With ice-bath cooling and inert gas atmosphere, 386 μl (4.43 mmol) of morpholine in 10 ml of dichloromethane are added dropwise to the reaction mixture within one hour. Subsequently, the yellow mixture is stirred for a further hour at 0 ° C, wherein a solid precipitates. It is then stirred for a further hour at room temperature and the mixture allowed to crystallize out at 4 ° C overnight. The reaction mixture is concentrated on a rotary evaporator to dryness. This gives 1.44 g of a bright yellow solid. Preparation of 5- (morpholine-N-carbonyl) -2-thioxo-1,3-dithiol-4-carboxylic acid chloride 5

200 mg (0.69 mmol) of morpholine amide 4 are placed in an argon-filled flask. There are added 3.00 ml (41.4 mmol) of thionyl chloride as reactant and solvent. The reaction mixture is heated with stirring to T = 30 ° C. After 1 h, the initially light yellow suspension turns into a clear orange solution. During the reaction, a steady evolution of gas can be observed. After a reaction time of 17 h, the excess thionyl chloride is completely removed in vacuo (1 mbar for 6 h). This gives 230 mg of an orange and viscous substance, which can not be further characterized due to their high reactivity and is thus used directly for subsequent reactions. Preparation of tert-butyl bis {3- [4- (morpholine-N-carbonyl) -2-thioxo-1,3-dithiol-5-carboxamido] propyl} carbamate 7

233.8 mg (0.75 mmol) of acid chloride 5 are dissolved in 4 ml of anhydrous dichloromethane and placed in the flask under argon atmosphere. Further, 105 .mu.l (0.75 mmol) of triethylamine in 1 ml of anhydrous dichloromethane at 0 to 5 ° C was added. This leads to a short smoke. There are 87.9 mg (0.38 mmol) dissolved in 4 ml of dry dichloromethane tert-butyl bis (3-aminopropyl) carbamate 6 slowly added dropwise to the reaction mixture over a period of 5 min. Subsequently, the yellow-brown solution is stirred for 1 h at 0 to 5 ° C, the ice bath removed and stirred for 4 days at RT. The solvent is removed by vacuum distillation leaving a brown oily crude product. This is taken up in a little chloroform and purified by column chromatography on silica gel 60 with the eluent mixture chloroform / propan-2-ol (9.5: 0.5). This gives 96 mg of the target product 7 as a yellow-brown oil. N, N '- (iminodipropane-3,1-diyl) bis [5- (morpholin-4-ylcarbonyl) -2-oxo-1,3-dithio-4-carboxamide] 8

81 mg (0.1 mmol) of compound 7 are dissolved in 4-5 ml of acetic acid. 199 mg of mercuric acetate (0.6 mmol) dissolved in 3-4 ml of acetic acid are added to the orange-clear solution, a white precipitate forming instantaneously. After stirring for 4 days at room temperature, the resulting suspension is concentrated to dryness. The product 8 is extracted by extracting 3-4 times with chloroform from the present solid and is present after removal of the solvent as a light yellow powder. N, N '- (iminodipropane-3,1-diyl) bis [4- (morpholin-4-yl) -4-oxo-2,3-disulfanyl-but-2-enamide] 9

0.1 mmol of compound 8 are dissolved in 4 ml of methanol. 0.4 mmol of NaOH, dissolved in 3 ml of methanol, are added to the yellow solution. After stirring at room temperature overnight, the resulting suspension is concentrated to dryness and the remaining solid is suspended with 5 ml of 1 mol / l hydrochloric acid. The product 9 is extracted by extracting 3-4 times with chloroform and is present after removal of the solvent as a light brown powder. Tin (IV) complex (10) of tetrathiol ligand 9

0.1 mmol of compound 9 are dissolved in 3 ml of acetonitrile. After addition of 0.1 mmol of SnCl ₄ , dissolved in 2 ml of acetonitrile, it is stirred for 4 hours at room temperature. After deduction of the solvent that remains Target product 10 as a pale yellow solid. Oxorhenium (V) Complex of Tetrathiol Ligand 9

At room temperature, 0.15 mmol of [NBu ₄ ] [ReOCl ₄ ] are initially charged in 100 ml of dry MeOH. With stirring, 0.15 mmol 9 dissolved in 100 ml of MeOH are slowly added to the reaction mixture. The color of the solution changes from green to orange-brown. The reaction is monitored by TLC and is complete after 1-2 h. The solvent is removed in vacuo, giving 190 mg of crude product as a brown oil. The oily product is purified over silica gel with an isopropanol / chloroform / ammonia / eluent mixture (5: 4: 1). ¹⁸⁸ Tetrathiol ligand ReO complex 9

0.1 mg of complex 10 dissolved in a mixture of 0.5 ml of methanol and 0.2 ml of propylene glycol is mixed with 0.5 ml of perrhenate generator eluate. After addition of 1.0 mg of SnCl ₂ , dissolved in 0.1 ml of 0.1 mol / l HCl, the reaction mixture is kept at 50 ° C. for 30 minutes. Radiochemical yield about 90%. ^99m tetrathiol ligand TCO complex 9

0.1 mg of complex 10 dissolved in a mixture of 0.2 ml of methanol, 0.2 ml of propylene glycol and 0.02 ml of 1N NaOH is mixed with 0.2 ml of pertechnetate generator eluate. After addition of 0.5 mg SnCl ₂ , dissolved in 0.1 ml of methanol, keeping the reaction mixture for 10 min at room temperature. Radiochemical yield about 90%.

Conjugation of an oligonucleotide

Introduction of an activated linker

To represent the maleinimidfuktionalisiertem derivative 1.1 equimol. MPS (3-maleimidopropionic acid succinimidyl ester) dissolved in acetonitrile or DMF (dimethylformamide) also dissolved in acetonitrile or DMF and 2.0 equimol. Triethylamine added oxo ligand 8 is slowly added dropwise at room temperature. The reaction mixture is then stirred for 24 h at RT. After removal of the solvent, the remaining residue is worked on directly. Conjugation of the oligonucleotide

The conjugation of the 1-mercaptohexyl-modified 17mer (SEQ ID No. 1) is dissolved in a 20 mmol / l borate buffer (pH = 5). The ligand precursor 12 is also dissolved in a 2.5-fold excess in the borate buffer and slowly added dropwise at room temperature. The reaction time is about 12 h. The isolation of the product is carried out by adding ethanol (up to an ethanol / water ratio of 90:10). After centrifugation, the supernatant is removed and the product pellets dissolved in a little water. This is followed by another ethanol precipitation. This cleaning procedure is carried out a total of 3 to 4 times. Production of the tin complex

to Removal of the protecting groups from compound 13 will concentrate them Ammonia dissolved and at 50 ° C for 4 h touched. The reaction solution is complete concentrated. To isolate the free tetrathiol oligonucleotide construct can the above-described purification via ethanol precipitation be used. The production of the tin complex is carried out as described above.

Synthesis of the 12-crown-4-bearing tetrathiol ligand

1st stage:

1.11 g (5.43 mmol) of the anhydride 3 are dissolved in 20 ml of anhydrous dichloromethane. Then argon is passed over this solution and cooled to 0-5 ° C. 0.95 g (5.43 mmol) of 1-aza-12-crown-4 are dissolved in 10 ml of anhydrous dichloromethane and added dropwise to the anhydride solution over a period of 60 min. The mixture is then stirred for a further hour under ice bath cooling and then for two more hours at room temperature. Subsequently, the crude product is concentrated on a rotary evaporator and the remaining oil is mixed with n-hexane. The supernatant, pale yellow hexane fraction is accepted. This process is repeated several times. The remaining, dark yellow oil is carefully removed under reduced pressure residual solvent. The product is in the form of a yellow, foamy solid (2.1 g). 2nd stage:

200 mg (0.527 mmol) of the 12-crown-4-amide are mixed with a 60-fold excess of thionyl chloride. The result is a yellow suspension which is heated to 30 ° C and stirred for 18 h. During the reaction time, the suspension turns into an orange, clear solution. Continuous gas development can still be observed. The reaction mixture is concentrated on a rotary evaporator to dryness and used for pressure equalization (aeration) argon. The residual oil is removed by means of a high-vacuum pump (<0.5 mbar) the remaining thionyl chloride and gaseous by-products remaining in the oil and used for pressure equalization (aeration) argon. The product obtained is available as a clear, orange and highly viscous oil and is due to the enormous sensitivity to hydrolysis immediately fed to the follow-up reaction. 3rd stage:

The freshly prepared carboxylic acid chloride is dissolved in 4 ml of anhydrous dichloromethane and placed in the flask under argon atmosphere. Next 2 molar equivalents of component 6 and 8 molar equivalents of triethylamine are dissolved in 1 ml of anhydrous dichloromethane and added to the acid chloride at 0 to 5 ° C. This leads to a short smoke. There are 87.9 mg (0.38 mmol) dissolved in 4 ml of dry dichloromethane tert-butyl bis (3-aminopropyl) carbamate 6 slowly added dropwise to the reaction mixture over a period of 5 min. Subsequently, the yellow-brown solution is stirred for 1 h at 0 to 5 ° C, the ice bath removed and stirred for 2 days at RT. The solvent is removed by vacuum distillation leaving a brown oily crude product. This is taken up in a little chloroform and purified by column chromatography on silica gel 60 with the eluent mixture chloroform / methanol (19: 1). The target product is obtained in 70% yield as a yellow-brown and powdery solid. 4th stage:

The bridged and Boc-protected compounds are dissolved in 2-4 ml of trifluoroacetic acid. After 12 h, the excess trifluoroacetic acid is removed under high vacuum conditions (<0.5 mbar). The products are in the form of a powdery yellow solid. If the trifluoroacetic acid can not be completely removed (product is present as a viscous oil), this can be dissolved in a little dichloromethane and precipitated by adding diethyl ether. To avoid losses in yield, the supernatants (dichloromethane and ether) are removed on a rotary evaporator. All deprotections are nearly quantitative and the products are present as trifluoroacetate salts. 5th stage:

Of the respective ligand is dissolved in methanol (1 ml per 100 mg ligand). The methanol must be free of oxygen. This can be done by initiating of helium and subsequent ultrasound treatment become. The dissolved ligand becomes 4-5 molar equivalents sodium hydroxide in the form of a methanolic sodium hydroxide solution added. After 12 h reaction time, the solvent are removed and the ligand extracted with dichloromethane. By addition of TFA and subsequent removal of the Solvent and excess TFA the product is as a TFA salt in the form of a deep red solid in front.

Synthesis of the 18-crown-6-bearing tetrathiol league

1st stage:

1.19 g (5.84 mmol) of thio-anhydride 3 are dissolved in 20 ml of anhydrous dichloromethane. Then argon is passed over this solution and cooled to 0-5 ° C. 1.54 g (5.84 mmol) of 1-aza-18-crown-6 are dissolved in 10 ml of dry dichloromethane and the anhydride solution is added dropwise over a period of one hour. The mixture is then passed under ice-bath cooling for a further hour and then stirred for two more hours. Subsequently, the crude product is concentrated on a rotary evaporator. This process is repeated several times. The residual, dark yellow oil is carefully removed under reduced pressure residual solvent. The product is in the form of a yellow, foamy solid (2.73 g). 2nd stage:

200 mg (0.428 mmol) of the 18-crown-6-amide are mixed with a 60-fold excess of thionyl chloride. The result is a yellow suspension which is heated to 30 ° C and stirred for 18 h. During the reaction time, the suspension turns into an orange, clear solution. Continuous gas development can still be observed. The reaction mixture is concentrated on a rotary evaporator to dryness and used for pressure equalization (aeration) argon. The residual oil is removed by means of a high-vacuum pump (<0.5 mbar) the remaining thionyl chloride and gaseous by-products remaining in the oil and used for pressure equalization (aeration) argon. The product obtained is available as a clear, orange and highly viscous oil and is due to the enormous sensitivity to hydrolysis immediately fed to the follow-up reaction. 3rd stage:

The freshly prepared carboxylic acid chloride is dissolved in 4 ml of anhydrous dichloromethane and placed in the flask under argon atmosphere. Next 2 molar equivalents of component 6 and 8 molar equivalents of triethylamine are dissolved in 1 ml of anhydrous dichloromethane and added to the acid chloride at 0 to 5 ° C. This leads to a short smoke. There are 87.9 mg (0.38 mmol) dissolved in 4 ml of dry dichloromethane tert-butyl bis (3-aminopropyl) carbamate 6 slowly added dropwise to the reaction mixture over a period of 5 min. Subsequently, the yellow-brown solution is stirred for 1 h at 0 to 5 ° C, the ice bath removed and stirred for 2 days at RT. The solvent is removed by vacuum distillation leaving a brown oily crude product. This is taken up in a little chloroform and purified by column chromatography on silica gel 60 with the eluent mixture chloroform / methanol (19: 1). The target product is obtained in 70% yield as a yellow-brown and powdery solid. 4th stage:

The bridged and Boc-protected compounds are dissolved in 2-4 ml of trifluoroacetic acid. After 12 h, the excess trifluoroacetic acid is removed under high vacuum conditions (<0.5 mbar). The products are in the form of a powdery yellow solid. If the trifluoroacetic acid can not be completely removed (product is present as a viscous oil), this can be dissolved in a little dichloromethane and precipitated by adding diethyl ether. To avoid losses in yield, the supernatants (dichloromethane and ether) are removed on a rotary evaporator. All deprotections are nearly quantitative and the products are present as trifluoroacetate salts. 5th stage:

Of the respective ligand is dissolved in methanol (1 ml per 100 mg ligand). The methanol must be free of oxygen. This can be done by initiating of helium and subsequent ultrasound treatment become. The dissolved ligand becomes 4-5 molar equivalents sodium hydroxide in the form of a methanolic sodium hydroxide solution added. After 12 h reaction time, the solvent are removed and the ligand extracted with dichloromethane. By addition of TFA and subsequent removal of the Solvent and excess TFA the product is as a TFA salt in the form of a deep red solid in front.

⁶⁴ Cu marks:

10 μg of tetrathiol ligand 9, dissolved in a mixture of 0.1 ml of DMF and 0.1 ml of a 100 mM aminomum acetate buffer solution (pH = 6.5), mixed with 40 MBq of [ ⁶⁴ Cu] -CuCl ₂ , shaken extensively with a vortexer and shaken for 10 min at room temperature in an Eppendorf mixer. The radiochemical yield is about 95%.

⁶⁸ Ga markings:

10 μg of tetrathiol ligand 9, dissolved in a mixture of 0.1 ml of DMF and 0.1 ml of a 100 mM aminomum acetate buffer solution (pH = 6.5) with freshly eluted [ ⁶⁸ Ga] GaCl ₃ (10 MBq ) and shaken extensively with a vortexer. The complexation takes place in an Eppendorf mixer and is complete after 10 min. The radiochemical yield is about 85%.

Preparation of active ester derivatives

Hydroxysuccinimidyl ester of the morpholino derivative:

N, N'-bis- (3 - {[(5- (morpholin-4-carbonyl) -2-thioxo [1,3] dithiol-4-carbonyl] amino} propyl) hydroxysuccinimidyl ester

100 mg of the TFA salt of the boc-deprotected morpholino compound are dissolved in 5 ml of anhydrous dichloromethane and 42.0 μl of triethylamine are added. After 10 minutes, 18.0 mg of succinic anhydride are added. The completion of the reaction can be determined by thin layer chromatography (silica gel plates with methanol-chloroform 1:10 as eluent). Subsequently, 5 ml of a 0.1 N HCl solution are added and the resulting 2-phase system is stirred vigorously for 3-4 h. This is followed by phase separation and, after complete removal of dichloromethane, the desired intermediate is obtained Product from the organic phase. This intermediate is again dissolved in 2 ml of anhydrous dichloromethane and 2 ml of anhydrous acetonitrile, and 1.5 molar equivalents of disuccinimidyl carbonate are added. This is followed by the addition of 50 .mu.l of triethylamine. The completeness of the reaction can be followed by thin layer chromatography (silica gel plates with methanol-chloroform 1:10 as eluent). After completion of the reaction, 0.1 N HCl solution is added and stirred for 1 h. From the organic phase, the product can be isolated by complete removal of the solvent.

Hydroxysuccinimidyl ester of the 12-crown-4 derivative:

N, N'-bis- (3 - {[(5- (1,4,7,10,13-pentaoxa-16-aza-cyclooctadecane-16-carbonyl} 2-thioxo [1,3] dithiol-4 carbonyl] amino} propyl) hydroxysuccinimidyl ester

100 mg of the TFA salt of the boc-deprotected 12-crown-4 compound are dissolved in 5 ml of anhydrous dichloromethane and 32.6 ul Added triethylamine. After 10 minutes, the addition of 14.1 mg succinic anhydride. The termination of the reaction can by thin layer chromatography (silica gel plates with Methanol-chloroform 1:10 as eluant). In connection 5 ml of a 0.1 N HCl solution are added and the resulting 2-phase system stirred vigorously for 3-4 h. This is followed by a phase separation and after completion Removal of dichloromethane gives the desired Intermediate from the organic phase. This interconnect is again dissolved in 2 ml of anhydrous dichloromethane and 2 ml of anhydrous Acetonitrile dissolved and 1.5 molar equivalents of disuccinimidyl carbonate added. This is followed by the addition of 50 .mu.l of triethylamine. The completeness of the reaction can be determined by thin-layer chromatography (Silica gel plates with methanol-chloroform 1:10 as eluent) followed become. After completion of the reaction, 0.1N HCl solution added and stirred for 1 h. From the organic phase can remove the product by complete removal of the solvent to be isolated purely.

Conjugation of hydroxysuccinimidyl active esters with an oligonucleotide and subsequent cleavage the thiol protecting groups:

Morpholin-BS DNA Tetrathiol Ligand:

The active ester is dissolved in 20-fold excess to the DNA in 200 ul dichloromethane and placed in an Eppendorf vial (low-DNA binding). The oligonucleotide is dissolved in 200 μl of 85 mM phosphate buffer at pH = 7.0 and added to the dichloromethane solution. Subsequently, 100 .mu.l of DMF are added and brought to reaction for at least 10 d and intensive shaking (mixing of the two phases necessary). The reaction control can be monitored by HPLC using a column designed specifically for the separation of oligonucleotides and buffered mobile phase. After completion, the organic phase can be separated and thus excess ligand recovered. The watery, yellow-colored phase are all solvent removed and the thiol-protected DNA conjugate via HPLC purification isolated. This is dissolved to deprotect completely deionized water and brought to a pH of 9-10 by addition of a sodium hydroxide solution (Suprapur). The completion of the reaction is determined by HPLC. After lyophilization, the product is obtained by HPLC purification and lyophilization as a yellow, fluffy solid. 12-crown-4-BS DNA Tetrathiol Ligand:

Of the Active ester is added in 20-fold excess to the DNA in 200 ul Dichloromethane dissolved and in an Eppendorf vial (low DNA binding) submitted. The oligonucleotide is dissolved in 200 μl of 85 mM phosphate buffer dissolved at pH = 7.0 and the dichloromethane solution added. Subsequently, 100 .mu.l of DMF are added and for at least 10 d and intensive shaking (Mixing of the two phases necessary) reacted. The reaction control can be carried out by HPLC using a specially developed for the separation of oligonucleotides Pillar and buffered eluent are tracked. To Termination, the organic phase can be separated and thus excess Ligand to be recovered. The watery, yellow colored phase are removed from all solvents and the thiol-protected DNA conjugate via HPLC purification isolated. This will be complete for deprotection Dissolved deionized water and by adding a sodium hydroxide solution (Suprapur) brought to a pH of 9-10. The termination the reaction is determined by HPLC. After lyophilization if the product is yellowed by HPLC purification and lyophilization, obtained fluffy solid.

Exemplary steat composition for radiopharmaceutical labeling with ¹⁸⁸ Re

• 0.1 mg tetrathiol ligand,
• 1.0 mg of stannous chloride
• 5 mg oxalic acid,
• 5 mg of gamma-cyclodextrin,
• 15 mg ascorbic acid

Exemplary stain composition for radiopharmaceutical labeling with ^99m Tc

• 0.1 mg tetrathiol ligand,
• 1.0 mg of stannous chloride
• 5 mg sodium titrate
• 5 mg mannitol

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - US 5175256 P [0005]
• WO 05108330 A1 [0007]

Cited non-patent literature

• - Guhlke S, Beets AL, Oetjen K, Mirzadeh S, Biersack HJ, Knapp FF Jr. Simple new method for effective concentration of 188Re solutions from alumina-based 188W-188Re generator. J Nucl Med. 2000, 41 (7): 1271-8. [0002]

Claims (20)

Ligand of the formula

wherein (CH-R1) and (CH-R1 ') represent substituted or unsubstituted methylene bridges, the individual R1 and R1', and R3 and R3 'are each independently selected from hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups, m and n are positive integers, where m represents the number of groups (CH-R1 ') and n represents the number of groups (CH-R1), the sum of m and n ranging from 4 to 10, R2 and R2 are independently selected from hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted carboxyl groups, substituted or unsubstituted aryl groups or Z, X is a substituted or unsubstituted methylene bridge of the formula CHR4 or an unsubstituted amino group (NH) or substituted amino group (NZ), wherein Z is - (CH-R5) pA, wherein (CH-R5) represents a substituted or unsubstituted methylene bridge, and pe is an integer representing the number of groups (CH-R5), where p is in the range of 2 to 8, A represents a linkable moiety, each of R4 and each R5 being independently selected from hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups. Ligand according to claim 1, characterized in that R2 and R2 'are independently selected from COOH, ester groups and substituted or unsubstituted Acid amide groups. Ligand according to Claim 1 or 2, characterized A is selected from ester groups and acid amide groups, Isothiocyanate groups, thiol groups, hydroxyl groups and halogens. A conjugate of a ligand according to any one of claims 1 to 3 and an organic molecule or a biomolecule, wherein the coupling via X, R2 or R2 'takes place. A conjugate according to claim 4, wherein the organic molecule or biomolecule is selected from proteins, Sugars, lipids, nucleic acids, antibodies, Peptides, oligonucleotides, small organic molecules, preferably cytostatics. Metal complex of the formula

wherein (CH-R1) and (CH-R1 ') represent monosubstituted or unsubstituted methylene bridges, the individual R1 and R1', R2 and R2 ', R3 and R3', m and n, and X are each independently selected as in Claim 1, Y is a metal atom or a metal complex, preferably selected from ^99m TcO, ¹⁸⁶ ReO, ¹⁸⁸ ReO, ^99m TcN, ¹⁸⁶ ReN, ¹⁸⁸ ReN ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, Sn, Tc ³⁺ and Re ³⁺ Metal complex according to claim 6, characterized in that R2 and R2 'independently are selected from COOH, ester groups and substituted or unsubstituted acid amide groups. Metal complex according to claim 6 or 7, characterized A is selected from ester groups and acid amide groups, Isothiocyanate groups, sulfhydryl groups, amino groups, hydroxyl groups and halogens. A conjugate of a metal complex according to any one of the claims 6 to 8 and a biomolecule, the coupling via X, R2 or R2 'takes place. A conjugate according to claim 9, wherein the organic molecule or biomolecule is selected from proteins, Sugars, lipids, nucleic acids, antibodies, Peptides, oligonucleotides and small organic molecules, preferably cytostatics. Use of a ligand according to any one of the claims 1 to 3 or a conjugate according to claim 4 or 5 for the preparation a metal complex. Use of a ligand according to any one of the claims 1 to 3 or a metal complex according to one of the claims 6 to 8 for coupling to biomolecules or organic molecules. A process for preparing a ligand according to any one of claims 1 to 3, comprising the following steps: a.) Reacting

with thionyl chloride, PCl ₃ , PCl ₅ or POCl ₃ to:

and possibly implementing

with thionyl chloride, PCl ₃ , PCl ₅ or POCl ₃ too

b.) reacting the acid chloride obtained in step a.) with R3- (NH) - (CH-R1) _n -X- (CH-R1 ') _m - (NH) -R3'

c.) Sulfur substitution by oxygen too

d.) hydrolysis to

Method according to claim 13, characterized in that that the preparation of the starting compound by a.) Implementation of dialkylacetylenedicarboxylate with ethylenetrithiocarbonate to 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid dialkyl ester, b.) Hydrolysis of the diester to the dicarboxylic acid, c.) condensation the dicarboxylic acid to the anhydride, d.) Implementing the so obtained 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid anhydride with an amine or alcohol. Process for the preparation of a radioactively labeled Metal complex or conjugate, by reacting a ligand one of claims 1 to 3, a conjugate according to one of Claims 4 or 5, a complex according to any one of the claims 6 to 8 with Y selected from Sn and / or a conjugate according to one of claims 9 or 10 selected with Y. from Sn, with pertechnetate, perrhenate in the presence of a reducing agent and optionally a nitrogen source, preferably a carbazate. Containing a kit for the preparation of a conjugate a.) a ligand according to any one of claims 1 to 3 and / or a complex according to any one of claims 6 to 8 selected with Y. made of Sn, b.) Coupling reagents, c.) buffer substances, d.) optionally stabilizers are preferably selected from Tartrate, oxalic acid and, ascorbic acid and optionally Excipients preferably selected from glucose, mannose, Mannitol and gamma-cyclodextrin. A kit for the preparation of a metal complex or conjugate comprising a.) A ligand according to any one of claims 1 to 3, a conjugate according to any one of claims 4 or 5, a A complex according to any one of claims 6 to 8 wherein Y is selected from Sn and / or a conjugate according to any one of claims 9 or 10 with Y selected from Sn, b) reducing agent, optionally a nitrogen source, preferably a carbazate, c.) Buffering agents, d optionally) stabilizers, preferably selected from tartrate, oxalic acid and ascorbic acid and e.) optionally auxiliary stool, preferably selected from glucose, mannose, mannitol and gamma-cyclodextrin Use of a ligand according to any one of the claims 1 to 3, a conjugate according to any one of claims 4 or 5, a metal complex according to any one of claims 6 to 8 or a conjugate according to any one of claims 9 or 10 or a kit according to claim 16 or 17 in nuclear medicine Diagnostics. Use of a ligand according to any one of the claims 1 to 3, a conjugate according to any one of claims 4 or 5, a metal complex according to any one of claims 6 to 8 or a conjugate according to any one of claims 9 or 10 or a kit according to claim 16 or 17 in endoradione nuclide therapy. Use of tin for the stabilization of tetrathioligands.