EP0696207A1

EP0696207A1 - Use of cyclopentadienylcarbonyl transition metal carboxylic acids and their derivatives for labelling proteins

Info

Publication number: EP0696207A1
Application number: EP94915087A
Authority: EP
Inventors: Martin Wenzel
Original assignee: Institut fuer Diagnostikforschung GmbH
Current assignee: Bayer Pharma AG
Priority date: 1993-04-22
Filing date: 1994-04-20
Publication date: 1996-02-14
Also published as: JPH08508985A; DE4313670A1; WO1994023757A1; CA2160979A1

Abstract

Radioactive metallocene carboxylic acids and their derivates having formula (I), in which R?1, R2, R3, R4 and R5¿ have different meanings and M stands for a radioactive metal isotope of the elements having the atomic numbers 21-29, 31, 42-44, 49 or 57-83, are used for radioactively labelling NH-active compounds. Also disclosed is a process for labelling said compounds, as well as new metallocene carboxylic acid derivates having formula (I) in which M stands for a Cr-51, Mn52, Ru-97, Ru-103, Re-186, Re-188, and Ir-191 isotope.

Description

Use of cyclopentadienylcarbonyl transition metal carboxylic acids and their derivatives for labeling proteins

The invention relates to the subject matter characterized in the claims, that is to say the use of radioactive metallocene carboxylic acids and their derivatives and methods for the radioactive labeling of proteins and other NH-active compounds. The invention further relates to new metallocene carboxylic acids and carboxylic acid derivatives of Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188 or Ir-191.

Radioactive labeled compounds are widely used in many areas of technology. In the field of medicine, radioactively labeled substances - such as labeled proteins - used for both diagnostic and therapeutic purposes. A known method of labeling proteins is that chelating agents are bound to proteins or oligopeptides in such a way that they do not lose their chelating property if possible. Corresponding chelating agents can be, for example, ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The actual labeling is then carried out by reaction with radioactive ions such as radioactive indium or technetium isotopes.

A disadvantage of using chelating agents for binding the radioactive isotopes is that the biological function of the protein can be changed by the chelating agent. In addition, part of the radioisotope that is primarily bound to the protein can be lost again by "re-chelating" by the body's complexing agents. However, the permanent binding of radioisotopes is the decisive prerequisite for radio-immunoassays - here the binding to proteins such as immunoglobulins - or a nuclear medicine application.

Protein labeling has also been carried out using iodine-labeled esters of N-hydroxysuccinimide (AEBolton, WM Hunter, Biochem. J., 133, 529, 1973). However, radioactive iodine isotopes are poorly suited for use in nuclear medicine for various reasons. Iodine-125 has too low an energy and a too long half-life, iodine-131 leads to a high radiation exposure due to its particle radiation and the iodine-123 is too expensive because its relatively short half-life requires extensive logistics. Furthermore, the structure of the protein can also be partially changed during iodination in such a way that the biological functions of the protein are only partially preserved. From WO 91/18908 ⁹⁹ Tc-cyclopentydienyl-carbonyl complexes with different side chains are known. The complexes described there are notable for high in vivo stability, so that "re-chelating" by the body's own complexing agents is not observed. The enrichment behavior determined by the side chain is also only slightly influenced by the complexing agent (cyclopentadienyl ring). However, it can be seen from the production process disclosed in WO 91/18908 that for each Tc complex (with a specific side chain - adapted to the respective diagnostic question), the corresponding cyclopentadienyl iron, chromium or cobalt complex is first synthesized or must be provided, which is then converted into the corresponding Tc complex by metal exchange reaction. This means that each individual 99mχ _c _mdd requires a separate preliminary stage. Another disadvantage of the method disclosed in WO 91/18908 is that it is not suitable for producing labeled, thermally unstable compounds.

It was therefore an object of the present invention to provide a class of substances which permit radioactive labeling of any amino group-containing molecules which show a specific in vivo enrichment behavior, and a method for labeling these molecules.

This object is achieved by the use of metallocene carboxylic acids and their derivatives of radioactive metal isotopes of the general formula I

R

^{M *M *}

R 2

⁽ D

wherein R ¹ , R ² , R ³ each represent a CO group or together represent a cyclopentadienyl radical which is optionally substituted by R ⁵ ', R ^{4 represents} a hydrogen atom, a C _r C ₁₀ alkyl radical or a group

-CO-CH ₃ , R ⁵ , R ⁵ 'independently of one another are -CO- (CH ₂ ) _m -COR ⁶ , - (CH ₂ ) _n -COR ⁶ , -CH = CH- (CH ₂ ) _n -COR ⁶ or -C≡C- (CH ₂ ) _n -COR6, where m for the digits 1 to 6, n for the digits 0 to 6, R ⁶ for chlorine, a group -NH-NH2, a radical -R ⁸ , -OR ⁷ or

-SR ⁷ is, with R ⁷ meaning hydrogen, a C ₁ -C ₁₀ alkyl,

R ⁸ in the meaning given for R ⁷ with the exception of hydrogen and a Cj-Ci _Q alkyl radical and M is a radioactive metal isotope of an element of atomic numbers 21-29,

31, 42-44, 49 or 57-83 means solved.

Using the compounds of formula I (hereinafter also referred to as "marker molecules") it is possible to convert any basic group-carrying molecules, in particular biomolecules - such as proteins, proteohormones or polypeptides - into stable, radioactively labeled substances which are both in the Radioimmune diagnostics as well as in nuclear medicine - for example for the in vivo display of receptors or antibody-binding structures, especially for the localization of tumors - can be used.

In addition, the metallocene carboxylic acids of the formula I are also suitable for introducing positron emitters into proteins.

Suitable radicals R ¹ , R ² , R ³ are in each case a carbonyl group or a pentahapto-bonded - if desired R ^{5 '} substituted - second cyclopentadienyl ring, the carbonyl groups being preferred. The radical R ⁴ is a -CO-CH ₃ group, a straight-chain or branched-chain Cj-CjQ-alkyl radical such as, for example, a methyl, ethyl, propyl, isopropyl, buty-1, isobutyl, tert. -Butyl, isopentyl, neopenty-1, hexyl or in particular a hydrogen atom.

Preferred radicals R ⁵ according to the invention are a -COOH -, a -CO- (CH2) 2-COOH - group, but in particular activated acid groups such as, for example, a -COC1 -, a -CO- (CH ₂ ) ₂ -CO-R ⁸ -, a -CO- (CH ₂ ) ₂ -COOR ⁸ -, a -CO-OR ⁸ - or a -CO-R ⁸ group with R ⁸ in the meaning of a succinimide or phthalimide radical.

The free carboxylic acid groups can, however, also be activated by other methods known to the person skilled in the art, for example by acid anhydride formation, which takes place with elimination of water by dimerization of two (intermolecular) carboxylic acid groups originating from different molecules. In the event that R ¹ , R ² , R ^{3 represent} a pentahapto-bonded second cyclopentadienyl ring which also contains a carboxylic acid group in the substituent R ^{5 '} , the anhydride formation can also take place intramolecularly.

The radioactive metal ions M used are preferably Mn-52, Tc-99m, Re-186, Re-188, Fe-52, Ru-95, Ru-97, Ru-103, Ir-191, including in particular Tc-99m, Ru-97 and Re-168.

The invention further relates to a method for the radioactive labeling of molecules containing basic groups using complexes of the formula I. The labeling is generally carried out in two stages. In the first step, the corresponding cyclopentadienl complexes of the formula II are first - analogously to the methods known to the person skilled in the art (see, for example, WO 91/18908 and examples 1-4) - in a metal exchange reaction

wherein R ¹ , R ² , R ³ , R ⁴ and R ^{5 have} the meanings given, but M ¹ is a non-radioactive isotope of the elements iron, cobalt or chromium, with metal oxides or metal salts of the desired metal ions of the elements of the atomic numbers 21-29, 31, 42-44, 49 or 57-83 implemented.

The radioactive compound of the formula I is generally purified by means of thin layer chromatography or HPLC. The localization of the compounds on the plates and the determination of the yield are carried out via radioactivity measurements.

In cases of longer-lived isotopes, e.g. Ru-97, this implementation can take place outside the clinic at a corresponding radiopharmaceutical manufacturer. In the case of short-lived isotopes, e.g. Tc-99m, this conversion would have to be carried out immediately before the application by the attending physician, with appropriate kits (analogous to those disclosed in WO 91/18908) being available.

The corresponding starting compounds of formula II with M ¹ in the meaning of iron, cobalt or chromium are commercially available in some cases or can be as described in M. Tanaka, J. Chromatogr. 292 (1984) 410 or Gmelin's Handbuch der

Inorganic chemistry [(Springer Verlag; Eighth Edition) volumes Ferrocene 2-4, 7, and 8 (part A)] described (or analogously to the compounds described there) can be easily prepared.

In the second reaction step, the actual labeling of the desired molecule follows. In this case, any substance which can be selected from NH groups, such as a protein, a proteohormone or a polypeptide, is reacted with the (the) radioactive metallocene carboxylic acid (derivative) of the formula I, if appropriate with the addition of an activator. The implementation is analogous to the methods described in Analytica Chimica Acta, 1992, 2, 145-159. Examples of activators for in situ activation of the carboxylic acid groups are dicyclohexylcarbodiimide (DDC), alkoxyacethylene, isobutylchlorocarbonate, benzotriazol-1-yl-tetramethyluronium tetrafluorate (TBTU). Bodanszky et. al., Principles of peptide synthesis, 1984.

In the case of metallocenes in which the carboxylic acid groups have already been activated, e.g. Succinimide ester, ester, thioester, amide or as an anhydride, the addition of an activator can be dispensed with.

The second reaction step takes place quickly and in good radiochemical yields, even at temperatures in the range from 20 to 40 ° C. and is usually carried out in aqueous buffer solutions, so that the conditions for routine use in the clinic are created.

The described “marker molecules” of the formula I and the method described above are suitable for the radioactive labeling of any NH-active compounds, such as proteins, immunoglobulins, antibodies, in particular antibodies against tumor tissue, Fab fragments,

Protein fragments, oligopeptides such as, for example, endothelin or partial endothelin sequences, amino acids, neurotransmitters, proteohormones, biogenic amines such as, for example, serotonin, histamine, γ-aminobutyric acid and derivatives of steroids and estrogenic steroids containing amino groups, amino sugars, ergoline compounds and other dopamine genes.

The compounds labeled using the metallocene carboxylic acids and carboxylic acid derivatives of the formula I according to the process mentioned can be used directly in in vivo diagnostics and therapy, the labeled compounds being formulated in a physiologically tolerable manner by the methods customary in galenics. This can e.g. by suspending or dissolving the respective compound in a physiologically compatible suspension medium such as water, electrolyte solutions, etc. respectively.

In the case of nuclear medical in vivo use, the marked

Compounds are generally dosed in amounts of less than 10 ^{** 10} mol / kg body weight, the exact dose depending on the region of the body being examined but in particular also on the examination method chosen in each case being able to vary widely. Based on an average body weight of 70 kg, the amount of radioactivity for diagnostic applications is between 180 and 1100 MBq, preferably 500-800 MBq per application. The application is usually intravenous, intraarterial, peritoneal or intratumoral, with intravenous application being preferred. Generally 0.1 to 2 ml of the agent in question are administered per test.

The choice of the respective radioisotope depends on the desired diagnostic method in which the radioactively labeled NH-active compounds are to be used.

The radio isotopes Tc-99m and Ru-97 are used in particular for the SPECT method. Ru-97 has the advantage over the short-lived Tc-99m that (due to its longer half-life of 2.88 days) it is already available as a finished "marker molecule" - for example in the form of the metallocene acid chloride or preferably as the N-succinimide ester. can be delivered to the clinics.

The isotopes Re-186 or Re-188 are used in particular for the labeling of proteins for radiation therapy.

Metallocenecarboxylic acids labeled with Fe-52, Mn-52 or Ru-95 are suitable for introducing positron emitters into proteins.

Some of the metallocenecarboxylic acids and carboxylic acid derivatives used according to the invention have not yet been described. The invention therefore also relates to new metallocene carboxylic acids and derivatives of the general formula I.

wherein R ¹ , R ² , R ³ each represent a CO group or together represent a cyclopentadienyl radical which is optionally substituted by R ^{5 '} , R ^{4 represents} a hydrogen atom, a C 1 -C ₁₀ -alkyl radical or a group

-CO-CH3, R ⁵ , R ⁵ 'independently of one another are -CO- (CH ₂ ) _m -COR ⁶ , - (CH ₂ ) _n -COR ⁶ , -CH = CH- (CH ₂ ) _n -COR ⁶ or -C≡C- (CH ₂ ) _n -COR ⁶ , where m for the digits 1 to 6, n for the digits 0 to 6, R ⁶ for chlorine, a group -NH-NH2, a radical -R ⁸ , -OR ⁷ or

-SR ⁷ , with -R ⁷ and -R ⁸ mean a succinimide or phthalimide residue and M is a Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188 or a Ir-191 stands.

The marker molecules of the formula I are distinguished in particular by the fact that they

do not or only marginally influence the biological activity of the labeled molecule, show high stability in vivo and "umchelation" does not occur, can be used universally for labeling any compounds containing NH groups, and that they contain NH groups containing under mild reaction conditions Connections react.

The method according to the invention is characterized in particular by the fact that the marking step takes place quickly and almost quantitatively under mild conditions. The method is therefore easy to carry out in the clinic on the one hand, but on the other hand it also enables the labeling of molecules, in particular those that are not particularly thermally stable, such as e.g. Proteins. Such molecules would not be radiolabelable using other known methods (see e.g. WO 91/18908).

The following examples serve to explain the subject matter of the invention in more detail, without wishing to restrict it to them.

"Cyclopentadienylcarbonyl-Technetium compounds" are hereinafter referred to as "Cytectrene". "Cyclopentadienylcarbonyl-Re compounds" are referred to as "Cyrhetrene" and "Rutheniumdicyclopentadienyl compounds" as "Ruthenocene" designated. Examples 1-4 describe the synthesis of various "marker molecules" used according to the invention, Examples 5-7 describe the derivatization (activation) of some "marker molecules" containing carboxylic acid groups, Examples 8-13 describe the radioactive labeling of various NH-active compounds.

Metal exchange reaction

Example 1 Tc-99m Cytectrencarboxylic Chloride

3 mg of ferrocene-carboxylic acid chloride, 0.5 mg of Mn (CO) ₅ Br and 20 μCi of radioactive Tc-99m pertechnetate, dissolved in 0.5 ml of dried methyl ethyl ketone, are placed in a small glass ampoule. The ampoule is melted off and heated for 1 h

150 ° C. Then the radioactive acid chloride is separated

Thin layer chromatography. The eluent is methylene chloride.

Rp ('cytctrenecarboxylic acid chloride'): 0.72 Rp (ferrocene carboxylic acid: 0.64

Radiochemical yield: 50%

Example 2 Tc-99m-cytctrenecarboxylic acid 1 mg ferrocenecarboxylic acid and 0.8 mg Mn (CO) ₅ Br are mixed with 10-20 MBq Tc-99m pertechnetate in 50μl tetrahydrofuran, melted in a glass ampoule under reduced pressure and 1 h at 170 ° C. heated. In order to separate the cytctrenecarboxylic acid from the residues of unreacted ferrocenecarboxylic acid, the contents of the ampoule are applied in a line to an ICW silicone rapid plate F 254 and chromatographed in methylene chloride / methanol (90:10). The cytctrenecarboxylic acid is eluted with 2-3 ml of chloroform / methanol (1: 1) and the eluate is evaporated to dryness under a nitrogen atmosphere. Rp (cytctrenecarboxylic acid '): 0.30 Rp (ferrocenecarboxylic acid'): 0.64 Radiochemical yield: 91%

Example 3 Tc-99m-Cytectren-CO- (CH ₁ ) _τ COOΗ.

2 mg ferrocenoyl propionic acid and 0.5 mg Mn (CO) ₅ Br are weighed into a small ampoule. Add 10 μl pertechnetate solution (physiological NaCl solution) with 50MBq Tc-99m and rinse with 50 μl tetrahydrofuran. After the ampoule has melted, the mixture is heated to 150 ° C. for 40 minutes. The purification is carried out by chromatography in cyclohexane / ether / glacial acetic acid (65: 35: 5). The Tc-99m cytctrenoyl propionic acid is eluted with 1 ml of tetrahydrofuran. RF (Ferrocenoyl propionic acid. 0.23

RF (Tc-99m-Cytectrenoylpropionic acid '): 0.17 Radiochemical yield: 79% Example 4 Ru-103-ruthenocenecarboxylic acid

In a glass ampoule, 2 mg of ferrocene carboxylic acid and a solution of 2 MBq

Ru-103-Cl3 melted in 50 μl dioxane with 3% HC1. The contents of the ampoule are heated to 130 ° C. for 1 h and then applied to thin-layer silica gel plates.

Chromatography in methylene chloride / methanol (88:12). After chromatography, a yellow band is obtained at an Rp = 0.64 and a non-colored, UV-active band at Rp = 0.56, which contains the radioactive ruthenocenecarboxylic acid. This radioactive peak is scraped off and eluted with chloroform / methanol (1: 1). Rp (ferrocene carboxylic acid: 0.64 Rp (ruthenocenecarboxylic acid '): 0.56 Radiochemical yield: 65%

Derivatization (activation)

Example 5 Tc-99m-Cytectrencarboxylic acid N-s ccinimide ester

The cytctrenecarboxylic acid obtained from Example 2 is taken up in 200 μl of tetrahydrofuran and reacted at room temperature with 0.25 mg of N-hydroxysuccinimide and 0.5 mg of dicyclohexylcarboxylic acid diimide for 30 minutes with stirring. The

Purification is carried out by thin layer chromatography in methylene chloride / methanol

(98: 2).

Rp (N-succinimide ester): 0.57

Radiochemical yield: 92%

Example 6 Tc-99m-Cytectrenoylpropionsä re-N-succinimide ester 200μl eluate of the Tc-99m-Cytectrenoylpropionic acid from Example 3, 4 mg of N-hydroxysuccinimide and 8 mg of dicyclohexylcarboxylic acid diimide are left under repeated shaking at room temperature. After 2 h the N-succinimide ester is obtained by chromatography in 79% yield. The purification is carried out by thin layer chromatography in methylene chloride / methanol (95: 5). Rp (N-succinimide ester '): 0.65 Radiochemical yield: 79% Example 7 Ru-103-ruthenocenecarboxylic acid N-succinimide ester The eluate of the radioactive ruthenocenecarboxylic acid obtained in Example 4 is evaporated to dryness, 5 mg of N-hydroxysuccinimide and 10 mg of N, N-dicyclohexylcarbodiimide in 300 μl of tetrahydrofuran are added. The mixture is then stirred at room temperature for 1 h. The ruthenocenecarboxylic acid N-succinimide ester is obtained after thin layer chromatography in methylene chloride / methanol (88:12) 95% yield. Rp (ruthenocenecarboxylic acid ^' ): 0.49 Rp (ruthenocenecarboxylic acid-N-succinimide ester): 0.82 Radiochemical yield: 95%

mark

Example 8 Tc-99m-Cytectrenoyl-glycine

The solution of the cytctrenecarboxylic acid N-succirimide ester obtained from Example 5 is concentrated to 10 .mu.l under a nitrogen atmosphere and mixed with 3 .mu.mol glycine - dissolved in 100 .mu.l borate buffer [0.1 molar, pH 8.5]. After stirring for 1 h at room temperature, the product is isolated by chromatography in ethanol and then purified by thin layer chromatography in chloroform acetone / formic acid (75: 20: 5).

Rp (Tc-99m-Cytectrenoyl-glvcin: 0.30 Radiochemical yield: 82%

Example 9 Tc-99m-Cytectrenoyl-glycine ethyl ester

10.93 MBq of Tc-99m-cytctrenecarboxylic acid prepared according to Example 2 are in 2 ml

Dissolved tetrahydrofuran. 0.2 ml of this solution are removed and 2 mg

Benzotriazol-l-yl tetramethyl uronium tetrafluorate and 1 ml of diisopropylethylamine. After shaking for 5 min at room temperature, this solution is added to 1 mg of glycine ethyl ester x HCl in 0.1 ml of tetrahydrofuran and 2 μl of diisopropylamine. The

The reaction mixture is stirred for 30 minutes. Thin layer chromatography is carried out in chloroform / acetone / formic acid (80: 2.5: 2.5).

R-p (Ferrocencarboxylic acid '): 0.39 R-p (Ferrocenovl-Glvcinethvlester'): 0.58

Rp (Tc-99m-Cytectrenoyl-Glvcinethv1ester '): 0.62

Radiochemical yield of Tc-99m-Cvtectrenoyl-G1vcinethy1estp.r- 98% Example 10 Tc-99m-Cytectrenoyl-N-glucosamine

0.76 mg (2 μmol) of HBTU, 1 μl of diisopropylamine and 100 μl of methylpyrrolidone are added to 9 MBq of Tc-99m labeled cytctrenecarboxylic acid. The reaction mixture is 30 min. stirred at room temperature and then applied to 20 cm wide TLC plates. In the eluent chloroform / ethanol (70:30), a radioactivity maximum of the desired glucosamine derivative is obtained at an Rp value of 0.40. After scraping off this Rp range, the radioactive substance is eluted with methanol. Re-chromatography in acetone / ethanol / ammonia (75: 20: 5) shows the radiochemical purity of this compound. Rp (Cytectrenoyl-N-glucosamiri): 0.40 Radiochemical yield: 80%

Example .11 Ru-103 ruthenocenoyl gfycin ethyl ester

To 1 MBq of the ruthenocenecarboxylic acid N-succinimide ester from Example 7, 1 mg of glycine ethyl ester and 100 μl of borate buffer (pH 9) are added after the solvent has been stripped off. After 1 h, 69% and after 2 h 91% of the desired ruthenocenoyl-glycine ethyl ester are obtained. The purification is carried out by thin layer chromatography in chloroform / acetone / formic acid (80: 2.5: 2.5). Rp (ruthenocenoyl-glycine ethyl ester): 0.43 Rp (ruthenocenecarboxylic acid-N-hvdoxysuccinimidester '): 0.56 Radiochemical yield: 91%

Example 12 Ru-103-Ruthenocenσyl heptapeptide

In a small glass, 1 mg of endothelin derivative [heptapeptide; Gly-His-Leu-Asp-Ue-Ile-Trp-COOH] with 4.8 KBq Ru-103-ruthenocenecarboxylic acid N-succinimide ester (prepared according to Example 7) in 100 μl borate buffer (pH 9) at room temperature. After 90 min, the batch is dissolved in chloroform methanol / ammonia (50:45:10). Chromatographies :. Radiochemical yield: 62% Example 13 Tc-99m Cytectrenoyl heptapeptide

19 MBq of N-succinimide cytctrencarboxylic acid ester (prepared according to Example 5) in 0.1 ml of THF are mixed with 1 mg of endothelin derivative [heptapeptide; Gly-His-Leu-Asp-Ile-Ile-Trp-COOH] dissolved in 0.2 ml borate buffer (pH 9). After stirring for 180 min, the mixture is chromatographed in the eluent ethyl acetate / MeOH / NH3 (50:40:10). Rp Cytectrencarbonsäuresuccinimidester: 0.69 Rg (Cytectrenoyl-Heptapeptidl: 0.33 Radiochemical yield: 53.6%

Claims

1. Use of metallocene carboxylic acids and their derivatives of radioactive metal isotopes of the general formula I

(I)

wherein

R ¹ , R ² , R ³ each represent a CO group or together represent a cyclopentadienyl radical which is optionally substituted by R ⁵ ',

R ^{4 represents} a hydrogen atom, a Cj-C ₁₀ alkyl radical or a group -CO-CH ₃ , R5, R5 'independently of one another are -CO- (CH ₂ ) _m -COR ⁶ , - (CH ₂ ) _n -COR ⁶ , -CH = CH- (CH ₂ ) _n -COR ⁶ or -C≡C- (CH ₂ ) _n -COR ⁶ , where m for the digits 1 to 6, n for the digits 0 to 6, R6 for Chlorine, a group -NH-NH2, a radical -R ⁸ , -OR ⁷ or -SR ⁷ , with

R ⁷ in the meaning of hydrogen, a C -CiQ- l yl-,

R ⁸ in the meaning given for R ⁷ with the exception of hydrogen and a C _j -C _j n-alkyl radical and M is a radioactive metal isotope of an element of atomic numbers 21-29,

31, 42-44, 49 or 57-83 means for the radioactive labeling of compounds containing NH groups.

2. Use of the compounds according to claim 1 for labeling proteins, immunoglobulins, antibodies, Fab fragments, protein fragments, oligopeptides, amino acids, histamine, γ-aminobutyric acid, neurotransmitters, proteohormones, biogenic amines, NH group-containing derivatives of steroids and estrogens Steroids, amino sugars, ergolines, and dopaminergic compounds.

3. Use of the compounds according to claim 1, in which the radioactive metal isotope is Mn-52, Tc-99m, Re-186, Re-188, Fe-52, Ru-95, Ru-97, Ru-103, Ir-191 .

4. Metallocene carboxylic acids and derivatives of the general formula I

wherein

R ¹ , R ² , R ³ each represent a CO group or together represent a cyclopentadienyl radical which is optionally substituted by R ^{5 '} , R ^{4 represents} a hydrogen atom, a C _r C ₁₀ alkyl radical or a group

-CO-CH3, R ⁵ , R ⁵ 'independently of one another are -CO- (CH ₂ ) _m -COR ⁶ , - (CH ₂ ) _n -COR ⁶ ,

-CH = CH- (CH ₂ ) _n -COR6 _or -C≡C- (CH ₂ ) _n -COR6, where m for the digits 1 to 6, n for the digits 0 to 6, R ⁶ for chlorine, one Group -NH-NH2, a radical -R ⁸ , -OR ⁷ or -SR ⁷ stands with -R ⁷ and -R ⁸ in the meaning of a succinimide or phthalimide residue and

M stands for a Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188 or an Ir-191.

5. metallocene carboxylic acid derivative according to claim 4, namely Ru-97-ruthenocenecarboxylic acid chloride.

6. Metallocene carboxylic acid derivative ^' according to claim 4, namely Ru-97-ruthenocenecarboxylic acid N-succinimide ester.

7. A process for the preparation of radioactively labeled NH-active, characterized in that a compound of formula I.

wherein R *, R ^, R ³ , R ⁴ and R-- ^{5 have} the meaning given, optionally with the addition of an activator, in a suitable solvent with an NH-active substance at 20 ° C - 40 ° C.

8. The method according to claim 7 wherein R-5 contains a -COC1 -, a succinimide or an N-hydroxysuccinimide residue.

9. The method according to claim 7, characterized in that the radioactive metallocene carboxylic acid derivative of the formula I Ru-97-ruthenocenecarboxylic acid-N-succinimide ester, Tc-99m-cytctrenecarboxylic acid-N-succinimide ester or a radioactive cyrhetrenecarboxylic acid-N-succinimide ester is used .

10. The method according to claim 7, characterized in that dicyclohexylcarbodumide or benzotriazol-1-yl-tetramethyl uronium tetrafluorate is used as the in situ activator.