CA2160979A1 - Use of cyclopentadienylcarbonyl transition metal carboxylic acids and their derivatives for labeling proteins - Google Patents
Use of cyclopentadienylcarbonyl transition metal carboxylic acids and their derivatives for labeling proteinsInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
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- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
- A61K51/0487—Metallocenes, i.e. complexes based on a radioactive metal complexed by two cyclopentadienyl anions
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Abstract
The invention relates to the use of radioactive metallocene carboxylic acids and their derivatives of formula I, (I) in which R1, R2, R3, R4 and R5 have different meanings, and M stands for a radioactive metal isotope of the elements of atomic numbers 21-29, 31, 42-44, 49 or 57-83, for radioactive labeling of NH-active compounds, a process for labeling the latter, as well as new metallocene carboxylic acid derivatives of formula I with M
meaning a Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188, Ir-191 isotope.
meaning a Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188, Ir-191 isotope.
Description
Use of Cyclopentadienylcarbonyl Transition Metal Carboxylic Acids and Their Derivatives for Labeling Proteins The invention relates to the object characterized in the claims, i.e., the use of radioactive metallocene carboxylic acids and their derivatives and process for 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 .
Radioactively-labled compounds are widely used in many fields of technology. In the field of medicine, radioactively-labeled substances -- such as, e.g., labeled proteins -- are used both for diagnostic purposes and for therapeutic purposes. A
known labeling process of proteins consists in that chelating agents are bound on proteins or oligopeptides, so that, as much as possible, they do not lose their chelating property.
Corresponding chelating agents can be, for example, ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The actual labeling then takes place by reaction with radioactive ions, such as, for example, radioactive indium or technetium isotopes.
A drawback in the use of chelating agents for bonding radioactive isotopes is that the biological function of the protein can be modified by the chelating agents. In addition, by "re-chelating" by endogenous complexing agents, a part of the radioisotope primarily bound on the protein can again be lost.
Radioactively-labled compounds are widely used in many fields of technology. In the field of medicine, radioactively-labeled substances -- such as, e.g., labeled proteins -- are used both for diagnostic purposes and for therapeutic purposes. A
known labeling process of proteins consists in that chelating agents are bound on proteins or oligopeptides, so that, as much as possible, they do not lose their chelating property.
Corresponding chelating agents can be, for example, ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The actual labeling then takes place by reaction with radioactive ions, such as, for example, radioactive indium or technetium isotopes.
A drawback in the use of chelating agents for bonding radioactive isotopes is that the biological function of the protein can be modified by the chelating agents. In addition, by "re-chelating" by endogenous complexing agents, a part of the radioisotope primarily bound on the protein can again be lost.
But the permanent bonding of the radioisotopes is the decisive requirement for radioimmunoassays -- here the bonding to proteins, such as, for example, immunoglobulins -- or for a nuclear medicine application.
Protein labeling has also been performed with the help of iodine-labeled esters of N-hydroxysuccinimide (A. E. Bolton, W.
M. Hunter, Biochem. J., 133, 529, 1973). Radioactive iodine isotopes are poorly suited, however, for nuclear medicine applications for various reasons. Iodine-125 has energy that is too low and a half-life that is too long, iodine-131 results in a high radiation exposure through its particle radiation and iodine-123 is too expensive, since its relatively short half-life requires extensive logistics. In addition, the structure of the protein can also be partially modified in the iodination, so that the biological functions of the protein are only just partially obtained.
~ mTc-Cyclopentyldienylcarbonyl complexes with different side chains are known from WO 91/18908. The complexes described there are distinguished by a high in vivo stability, so that a "re-chelation" by endogenous complexing agents is not observed.
Also, the concentration behavior determined by the side chain is only insignificantly influenced by the complexing agents (cyclopentadienyl ring). But it follows from the production process disclosed in WO 91/18908 that for each Tc complex (with side chain matched specifically to the respective diagnostic problem), the corresponding cyclopentadienyl-iron, -chromium- or -cobalt complex must first be synthesized or prepared, which then is converted to the corresponding Tc complex by metal exchange reaction. That is to say, every individual ~Tc compound requires its own precursor. Another drawback of the process disclosed in WO 91/18908 is that it is not suitable for the production of labeled, thermally unstable compounds.
The object of this invention was therefore a class of substances that allows radioactive labeling of any amino-group-containing molecules that show a specific in-vivo concentration behavior, as well as to prepare a process for labeling these molecules.
According to the invention, this object is achieved by the use of metallocene carboxylic acids and their derivatives of radioactive metal isotopes of general formula I
~ ~R
R ¦2 R
in which R1~ R2, R3 each stand for a C0 group or together mean a cyclopentadienyl radical, which optionally is substituted by Rs, R4 stands for a hydrogen atom, a C1-C10 alkyl radical or a group -C0-CH3, , R5, R5, independently of one another, stand for -CO-(CH2)m-COR6, -(CH2)n-COR6, -CH=CH-(CH2)n-COR6 or -C_C- (CH2) n--COR6, in which m stands for numbers 1 to 6, n stands for numbers O to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -oR7 or -SR7, with R7 meaning hydrogen, a Cl-C10 alkyl radical, O O
-N I or -N ~ radical, and R8 in the meaning indicated for R7 with the exception of hydrogen and a Cl-C10 alkyl radical and M means a radioactive metal isotope of an element of atomic numbers 21-29, 31, 42-44, 49 or 57-83.
By using the compounds of formula I (designated below also as "marker molecules"), it is possible to convert any molecules carrying basic groups, especially biomolecules -- such as proteins, proteohormones or polypeptides -- to stable, radioactively-labeled substances, which can be used both in radioimmunodiagnosis and in nuclear medicine -- for example for in vivo representation of receptors or antibody-bonding structures, especially for localizing tumors.
Moreover, the metallocene carboxylic acids of formula I are also suitable to introduce positron-emitters into proteins.
As radicals R1, R2, R3, respectively a carbonyl group or a pentahapto-bound -- optionally R5-substituted -- second cyclopentadienyl ring is suitable, the carbonyl groups being preferred.
As radical R4, a -CO-CH3 group, a straight-chain or branched-chain C1-C10 alkyl radical, such as, e.g., a methyl, ethyl, propyl, isopropyl, butyl-l, isobutyl, tert-butyl, isopentyl, neopentyl-l, hexyl radical or especially a hydrogen atom, are suitable.
According to the invention, preferred radicals R5 are a -COOH group, a -CO-(CH2)2-COOH group, but especially activated acid groups, such as, e.g., a -COCl group, a -CO-(CH2)2-CO-R8 group, a -CO-(CH2)2-COOR8 group, a -CO-OR8 group or a -CO-R8 group with R8 meaning a succinimide or phthalimide radical.
But the activation of the free carboxylic acid groups can also take place according to other methods known to one skilled in the art, thus, it takes place, e.g., by acid anhydride formation, during dehydration by dimerization of two carboxylic acid groups derived (in an intermolecular manner) from the different molecules. For the case that R1, R2, R3 stand for a pentahapto-bound second cyclopentadienyl ring, which also contains a carboxylic acid group in substituent R5', the anhydride formation can also take place in an intramolecular manner.
As radioactive metal ions M, Mn-52, Tc-99m, Re-186, Re-188, Fe-52, Ru-95, Ru-97, Ru-103, Ir-191, among them especially Tc-99m, Ru-97 and Re-168, are preferably used.
The invention further relates to a process for radioactive labeling of molecules that contain basic groups by using complexes of formula I. The labeling takes place generally in two stages.
In the first step -- analogously to the method known to one skilled in the art (see, e.g., W0 91/18908, as well as Examples 1-4) -- the corresponding cyclopentadiene complexes of formula II
R
l2 R
R (I~
in which R1, R2, R3, R4 and Rs have the indicated meanings, but M1 stands for a nonradioactive isotope of elements iron, cobalt or chromium, are first reacted in a metal exchange reaction with metal oxides or metal salts of the desired metal ions of elements of atomic numbers 21-29, 31, 42-44, 49 or 57-83.
The purification of the radioactive compound of formula I
takes place generally by thin-layer chromatography or HPLC. The localization of the compounds on the plates as well as the yield determinations take place by measurements of radioactivity.
, In cases of longer-lived isotopes, such as, e.g., Ru-97, this reaction can take place even outside clinical practice in the case of a suitable manufacturer of radiopharmaceutical agents. In the case of short-lived isotopes, such as, e.g., Tc-99m, this reaction would be performed directly before the administration by the attending physician, and the appropriate kits would have to be provided for (analogously to those disclosed in WO 91/18908).
The corresponding starting compounds of formula II with meaning iron, cobalt or chromium are partially obtainable commercially or can be easily produced as described in M. Tanaka, J. Chromatogr. 292 (1984) 410 or Gmelin's Handbuch der Anorganischen Chemie [Gmelin's Inorganic Chemistry Manual]
[(Springer Verlag; 8th Edition), Ferrocene Volumes 2-4, 7 and 8 (part A)~ (or analogously to the compounds described there).
The actual labeling of the desired molecule follows in the second reaction step. In this case, any selectable substance carrying NH groups, such as, e.g., a protein, a proteohormone or a polypeptide, is reacted with the radioactive metallocene carboxylic acid (derivative) of formula I, optionally by adding an activator. The reaction takes place analogously to the methods described in Analytica Chemica Acta, 1992, 264, 145-lS9.
As activators for in situ activation of the carboxylic acid groups, for example, dicyclohexylcarbodiimide (DDC), alkoxyacetylene, isobutyl chlorocarbonate, benzotriazol-l-yl-tetramethyl-uronium-tetrafluorate (TBTU), can be mentioned.
_ Other activators are described by Bodanszky et al., Principles of Peptide Synthesis, 1984.
In the case of metallocenes in which the carboxylic acid groups are present already in activated form, e.g., succinimide esters, esters, thioesters, amide or as anhydride, the addition of an activator can be dispensed with.
The second reaction step takes place quickly and in good radiochemical yields, already at temperatures in the range of 20 to 40C and is generally performed in aqueous buffer solutions, so that the requirements for a routine use in clinical practice are established.
The described "marker molecules" of formula I as well as the previously described process are suitable for radioactive labeling of any NH-active compounds, such as, for example, proteins, immunoglobulins, antibodies, especially antibodies against tumor tissue, Fab fragments, protein fragments, oligopeptides, such as, for example, endothelin or endothelin partial sequences, amino acids, neurotransmitters, proteohormones, biogenous amines, such as, for example, serotonin, histamine, y-aminobutyric acid as well as amino group-containing derivatives of steroids and estrogenic steroids, amino sugars, ergolines and other dopaminergic compounds.
The compounds labeled by using the metallocene carboxylic acids and carboxylic acid derivatives of formula I according to the mentioned process can be used directly in in vivo diagnosis and therapy, and the labeled compounds are formulated in a physiologically compatible way according to the methods usual in galenicals. This can take place, e.g., by suspending or dissolving the respective compound in a physiologically compatible suspension medium, such as water, electrolyte solutions, etc.
In in vivo use in nuclear medicine, the labeled compounds are generally dosed in amounts smaller than 10-10 mol/kg of body weight, and the exact dose can vary greatly as a function of the examined region of the body but especially also of the respectively selected method of examination.
Starting from 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 administration. Administration takes place normally intravenously, intraarterially, peritoneally or intratumorally, the intravenous administration being preferred. Generally 0.1 to 2 ml of the agent in question is administered per test.
The selection of the respective radioisotope depends on the desired diagnostic method, in which the radioactively-labeled NH-active compound is to be used.
In particular, radioisotopes Tc-99m and Ru-97 are used for the SPECT process. Relative to the short-lived Tc-99m, Ru-97 has the advantage that it can be supplied to clinics (based on its longer half-life of 2.88 days) already as a finished "marker molecule" -- for example, in the form of the metallocene acid chloride or preferably as N-succinimide ester.
Isotopes Re-186 or Re-188 are used especially in the labeling of proteins for radiation therapy.
Metallocene carboxylic acids labeled with Fe-52, Mn-52 or Ru-95 are suitable for the introduction of positron emitters into proteins.
Some of the metallocene carboxylic acids and carboxylic acid derivatives used according to the invention have not as yet been described. The invention therefore also relates to new metallocene carboxylic acids and derivatives of general formula I
~ R4 R
in which R1, R2, R3 each stand for a CO group or together mean a cyclopentadienyl radical, which optionally is substituted by R5, R~ stands for a hydrogen atom, a C,-C10 alkyl radical or a group -CO-CH3, R5, Rs, independently of one another, stand for -CO-- (CH2)~-COR6, -(CH2)n-COR6, -CH=CH-(CH2)n-COR6 or -C_C- (CH2) n-COR6, in which m stands for numbers l to 6, n stands for numbers O to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -oR7 or -SR7, with -R7 and -R8 meaning a succinimide or phthalimide radical and M stands for a Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188 or an Ir-l91.
The marker molecules of formula I are distinguished especially in that they -- do not or only insignificantly influence the biological activity of the labeled molecule, -- show a high stability in vivo and a "re-chelation" does not occur, -- can be used universally for labeling any compounds that contain NH groups and in that -- they react with compounds that contain NH groups under mild reaction conditions.
The process according to the invention is distinguished especially in that the labeling step occurs quickly and almost quantitatively under mild conditions. The process can therefore be performed easily in clinical practice, on the one hand, but also makes possible, on the other hand, the labeling of --especially thermally -- less loadable molecules, such as, e.g., proteins. Such molecules could not be radioactively labeled according to other known processes (see, e.g., W0 91/18908).
The following examples are used for a more detailed explanation of the object of the invention, without intending to be limited to this object. "Cyclopentadienylcarbonyl-technetium compounds" are designated below as "cytectrenes."
''CyClopentadienylcarbonyl-Re compounds~ are designated as "cyrhetrenes~ and ~ruthenium dicyclopentadienyl compounds" as "ruthenocenes.n Examples 1-4 describe the synthesis of various "marker molecules~ used according to the invention; Examples S-7 describe the derivatization (activation) of some "marker molecules" that contain carboxylic acid groups; Examples 8-13 describe the radioactive labeling of various NH-active compounds.
._ Metal Exchange Reaction Example ~ Tc-99m Cytectrene carboxylic acid chloride 3 mg of ferrocene carboxylic acid chloride, 0.5 mg of Mn(CO)5Br and 20 ~Ci of radioactive Tc-99m-pertechnetate, dissolved in 0.5 ml of dried methyl ethyl ketone, are added to a small glass ampoule. The ampoule is sealed off and heated for 1 hour to 150C. Then, the radioactive acid chloride is separated by thin-layer chromatography. Eluant is methylene chloride.
Rr (Cytectrene carboxylic acid chloride): 0.72 Rr (Ferrocene carboxYlic acid): 0.64 Radiochemical yield: 50%
Example 2 Tc-99m-Cytectrene carboxylic acid 1 mg of ferrocene carboxylic acid and 0.8 mg of Mn(CO)sBr are mixed with 10-20 MBq of Tc-99m-pertechnetate in 50 ~l of tetrahydrofuran, sealed in a glass ampoule under reduced pressure and heated for 1 hour to 170C. For separation of the cytectrene carboxylic acid from the radicals of unreacted ferrocene carboxylic ac d, the content of the ampoule is applied in streaks on an ICW-silicone rapid plate F 254 and chromatographed in methylene chloride/methanol (90:10). The cytectrene carboxylic acid is eluted with 2-3 ml of chloroform/methanol (1:1), and the eluate is evaporated to dryness under nitrogen atmosphere.
Rr (Cytectrene carboxylic acid): 0.30 Rr (Ferrocene carboxylic acid): 0.64 Radiochemical Yield: 91%
216097~
-Example 3 Tc-99m-Cytectrene-C0-(CH2)2-COOH
2 mg of ferrocenoylpropionic acid and 0.5 mg of Mn(C0)5Br are weighed into a small ampoule. 10 ~l of pertechnetate solution (physiological NaCl solution) with 50 MBq of Tc-99m is added to it and flushed with 50 ~l of tetrahydrofuran. After sealing off the ampoule, it is heated for 40 minutes to 150C.
The purification takes place by chromatography in cyclohexane/ether/glacial acetic acid (65:35:5). The Tc-99m-cytectrenoylpropionic acid is eluted with 1 ml of tetrahydrofuran.
Rr (Ferrocenoylpropionic acid): 0.23 Rr (Tc-99m-cytectrenoYlpropionic acid): 0.17 Radiochemical Yield: 79%
Example 4 Ru-103-ruthenocene carboxylic acid 2 mg of ferrocene carboxylic acid and a solution of 2 MBq of RU-103-C13 in 50 ~l of dioxane with 3% HCl are sealed in a glass ampoule. The contents of the ampoule are heated for 1 hour to 130C and then applied to silica gel thin-layer plates.
Chromatography in methylene chloride/methanol (88:12). After the chromatography, a yellow strip with an RF = - 64 and a non-colored, W -active strip with RF = . 56, which contains the radioactive ruthenocene carboxylic acid, are obtained. This radioactive peak is scraped off and eluted with chloroform/
methanol (1:1).
Rr (Ferrocene carboxylic acid): 0.64 Rr (Ruthenocene carboxylic acid): 0.56 Radiochemical yield: 65%
Derivatization ~Activation) Example 5 Tc-ssm-cytectrene carboxylic acid-N-succinimide ester The cytectrene carboxylic acid obtained from Example 2 is taken up in 200 ~1 of tetrahydrofuran and reacted at room temperature with 0.25 mg of N-hydroxysuccinimide as well as 0.5 mg of dicyclohexylcarboxylic acid diimide for 30 minutes with stirring. Purification takes place by thin-layer chromatography in methylene chloride/methanol (98:2).
Rr (N-Succinimide ester): 0.57 Radiochemical Yield: 92%
Example 6 Tc-99m-Cytectrenoylpropionic acid-N-succinimide ester 200 ~1 of eluate of the Tc-99m-cytectrenoylpropionic acid of Example 3, 4 mg of N-hydroxysuccinimide and 8 mg of dicyclohexylcarboxylic acid diimide are allowed to stand at room temperature with repeated shaking. After 2 hours, the N-succinimide ester is obtained by chromatography in 79% yield.
The purification takes place by thin-layer chromatography in methylene chloride/methanol (95:5).
Rr (N-Succinimide ester): 0.65 Radiochemical yield: 79%
Example 7 Ru-103-Ruthenocene carboxylic acia-N-suc¢inimide e~ter The eluate of the radioactive ruthenocene carboxylic acid obtained in Example 4 is evaporated to dryness, mixed with 5 mg of N-hydroxysuccinimide and 10 mg of N,N-dicyclohexylcarbodiimide in 300 ~l of tetrahydrofuran. Then, it is stirred for 1 hour at room temperature. The ruthenocene carboxylic acid-N-succinimide ester is obtained after thin-layer chromatography in methylene chloride/methanol (88:12) in 95% yield.
Rr (Ruthenocene carboxYlic acid): 0.49 Rr fRuthenocene carboxYlic acid-N-succinimide ester): 0.82 Radiochemical yield: 95~ -Labeling Example 8 Tc-99m-Cytectrenoyl-glycine The solution of cytectrene carboxylic acid-N-succinimide ester obtained from Example 5 is concentrated by evaporation under nitrogen atmosphere to 10 ~l and mixed with 3 ~mol of glycine -- dissolved in 100 ~l of borate buffer [0.1 molar, pH
8.5]. After 1 hour of stirring 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)-Rr (Tc-99m-Cytectrenoyl-glycine): 0.30 Radiochemical yield: 82%
17 216097~
Example 9 ~c-gsm-cytectrenoyl-glycine-ethyl ester 10.93 MBq of Tc-99m-cytectrene carboxylic acid produced according to Example 2 is dissolved in 2 ml of tetrahydrofuran.
0.2 ml is removed from this solution and mixed with 2 mg of benzotriazol-l-yl-tetramethyl-uronium-tetrafluorate and 1 ml of diisopropylethylamine. After S minutes of shaking at room temperature, this solution is added to 1 mg of glycine ethyl ester x HCl in 0.1 ml of tetrahydrofuran and 2 ~1 of diisopropylamine. The reaction mixture is stirred for 30 minutes. The thin-layer chromatography takes place in chloroform/acetone/formic acid (80:2, 5:2, S).
Rr (Ferrocene carboxylic acid): 0.39 Rr (Ferrocenoyl-glycine ethyl ester): 0.58 Rr (Tc-99m-Cytectrenoyl-glycine ethYl ester): 0.62 Radiochemical yield of Tc-99m-cytectrenoYl-glycine ethYl ester: 98%
Example 10 Tc-99m-Cytectrenoyl-N-glucosamine 0.76 mg (2 ~mol) of HBTU, 1 ~1 of diisopropylamine and 100 ~1 of methyl pyrrolidone are added to 9 MBq of Tc-99m-labeled cytectrene carboxylic acid produced according to Example 2. The reaction mix~ure is stirred for 30 minutes at room temperature and then applied to 20 cm-wide TLC plates. In the mobile solvent chloroform/e~hanol (70:30) at an RF of 0.40, a radioactivity maximum of t~e desired glucosamine derivative is obtained. After scraping off this RF area, the radioactive substance is eluted 18 216~979 -with methanol. Re-chromatography in acetone/ethanol/ammonia (75:20:5) shows the radiochemical purity of this compound.
Rr (CYtectrenoyl-N-glucosamine): 0.40 Radiochemical Yield: 80%
Example 11 Ru-103-Ruthenocenoyl-glycine-ethyl ester After removal of the solvent, 1 mg of glycine ethyl ester and 100 ~l of borate buffer (pH 9) are added to 1 MBg of the ruthenocene carboxylic acid-N-succinimide ester of Example 7.
After 1 hour, 69% is obtained, and after 2 hours, 91% of the desired ruthenocenoyl-glycine ethyl ester is obtained. The purification takes place by thin-layer chromatography in chloroform/acetone/formic acid (80:2, 5:2, 5).
Rr (RuthenocenoYl-glYcine-ethYl ester): 0.43 Rr (Ruthenocene carboxylic acid-N-hydroxYsuccinimide ester):
0.56 Radiochemical yield: 91%
~xample 12 Ru-103-Ruthenocenoyl-heptapeptide 1 mg of endothelin derivative [heptapeptide; Gly-His-Leu-Asp-Ile-Ile-Trp-COOH] with 4.8 KBq of Ru-103-ruthenocene carboxylic acid-N-succinimide ester (produced according to Example 7) is stirred in a small glass in 100 ~1 of borate buffer (pH 9) at room temperature. After 90 minutes, the batch is chromatographed in the mobile solvent of chloroform/methanol/
ammonia (50:4S:10).
Radiochemical Yield: 62%
19 216097~
Example 13 Tc-99m-Cytectrenoyl-heptapeptide 19 MBq of cytectrene carboxylic acid-N-succinimide ester (produced according to Example 5) in 0.1 ml of THF is mixed with 1 mg of endothelin derivative [heptapeptide; Gly-His-Leu-Asp-Ile-Ile-Trp-CoOH] dissolved in 0.2 ml of borate buffer (pH 9). After 180 minutes of stirring, the batch is chromatographed in the mobile solvent ethyl acetate/MeOH/NH3 (50:40:10).
Rr Cytectrene carboxYlic acid succinimide ester: 0.69 Rr (Cytectrenoyl-heptapeptide): 0.33 Radiochemical yield: 53.6%
Protein labeling has also been performed with the help of iodine-labeled esters of N-hydroxysuccinimide (A. E. Bolton, W.
M. Hunter, Biochem. J., 133, 529, 1973). Radioactive iodine isotopes are poorly suited, however, for nuclear medicine applications for various reasons. Iodine-125 has energy that is too low and a half-life that is too long, iodine-131 results in a high radiation exposure through its particle radiation and iodine-123 is too expensive, since its relatively short half-life requires extensive logistics. In addition, the structure of the protein can also be partially modified in the iodination, so that the biological functions of the protein are only just partially obtained.
~ mTc-Cyclopentyldienylcarbonyl complexes with different side chains are known from WO 91/18908. The complexes described there are distinguished by a high in vivo stability, so that a "re-chelation" by endogenous complexing agents is not observed.
Also, the concentration behavior determined by the side chain is only insignificantly influenced by the complexing agents (cyclopentadienyl ring). But it follows from the production process disclosed in WO 91/18908 that for each Tc complex (with side chain matched specifically to the respective diagnostic problem), the corresponding cyclopentadienyl-iron, -chromium- or -cobalt complex must first be synthesized or prepared, which then is converted to the corresponding Tc complex by metal exchange reaction. That is to say, every individual ~Tc compound requires its own precursor. Another drawback of the process disclosed in WO 91/18908 is that it is not suitable for the production of labeled, thermally unstable compounds.
The object of this invention was therefore a class of substances that allows radioactive labeling of any amino-group-containing molecules that show a specific in-vivo concentration behavior, as well as to prepare a process for labeling these molecules.
According to the invention, this object is achieved by the use of metallocene carboxylic acids and their derivatives of radioactive metal isotopes of general formula I
~ ~R
R ¦2 R
in which R1~ R2, R3 each stand for a C0 group or together mean a cyclopentadienyl radical, which optionally is substituted by Rs, R4 stands for a hydrogen atom, a C1-C10 alkyl radical or a group -C0-CH3, , R5, R5, independently of one another, stand for -CO-(CH2)m-COR6, -(CH2)n-COR6, -CH=CH-(CH2)n-COR6 or -C_C- (CH2) n--COR6, in which m stands for numbers 1 to 6, n stands for numbers O to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -oR7 or -SR7, with R7 meaning hydrogen, a Cl-C10 alkyl radical, O O
-N I or -N ~ radical, and R8 in the meaning indicated for R7 with the exception of hydrogen and a Cl-C10 alkyl radical and M means a radioactive metal isotope of an element of atomic numbers 21-29, 31, 42-44, 49 or 57-83.
By using the compounds of formula I (designated below also as "marker molecules"), it is possible to convert any molecules carrying basic groups, especially biomolecules -- such as proteins, proteohormones or polypeptides -- to stable, radioactively-labeled substances, which can be used both in radioimmunodiagnosis and in nuclear medicine -- for example for in vivo representation of receptors or antibody-bonding structures, especially for localizing tumors.
Moreover, the metallocene carboxylic acids of formula I are also suitable to introduce positron-emitters into proteins.
As radicals R1, R2, R3, respectively a carbonyl group or a pentahapto-bound -- optionally R5-substituted -- second cyclopentadienyl ring is suitable, the carbonyl groups being preferred.
As radical R4, a -CO-CH3 group, a straight-chain or branched-chain C1-C10 alkyl radical, such as, e.g., a methyl, ethyl, propyl, isopropyl, butyl-l, isobutyl, tert-butyl, isopentyl, neopentyl-l, hexyl radical or especially a hydrogen atom, are suitable.
According to the invention, preferred radicals R5 are a -COOH group, a -CO-(CH2)2-COOH group, but especially activated acid groups, such as, e.g., a -COCl group, a -CO-(CH2)2-CO-R8 group, a -CO-(CH2)2-COOR8 group, a -CO-OR8 group or a -CO-R8 group with R8 meaning a succinimide or phthalimide radical.
But the activation of the free carboxylic acid groups can also take place according to other methods known to one skilled in the art, thus, it takes place, e.g., by acid anhydride formation, during dehydration by dimerization of two carboxylic acid groups derived (in an intermolecular manner) from the different molecules. For the case that R1, R2, R3 stand for a pentahapto-bound second cyclopentadienyl ring, which also contains a carboxylic acid group in substituent R5', the anhydride formation can also take place in an intramolecular manner.
As radioactive metal ions M, Mn-52, Tc-99m, Re-186, Re-188, Fe-52, Ru-95, Ru-97, Ru-103, Ir-191, among them especially Tc-99m, Ru-97 and Re-168, are preferably used.
The invention further relates to a process for radioactive labeling of molecules that contain basic groups by using complexes of formula I. The labeling takes place generally in two stages.
In the first step -- analogously to the method known to one skilled in the art (see, e.g., W0 91/18908, as well as Examples 1-4) -- the corresponding cyclopentadiene complexes of formula II
R
l2 R
R (I~
in which R1, R2, R3, R4 and Rs have the indicated meanings, but M1 stands for a nonradioactive isotope of elements iron, cobalt or chromium, are first reacted in a metal exchange reaction with metal oxides or metal salts of the desired metal ions of elements of atomic numbers 21-29, 31, 42-44, 49 or 57-83.
The purification of the radioactive compound of formula I
takes place generally by thin-layer chromatography or HPLC. The localization of the compounds on the plates as well as the yield determinations take place by measurements of radioactivity.
, In cases of longer-lived isotopes, such as, e.g., Ru-97, this reaction can take place even outside clinical practice in the case of a suitable manufacturer of radiopharmaceutical agents. In the case of short-lived isotopes, such as, e.g., Tc-99m, this reaction would be performed directly before the administration by the attending physician, and the appropriate kits would have to be provided for (analogously to those disclosed in WO 91/18908).
The corresponding starting compounds of formula II with meaning iron, cobalt or chromium are partially obtainable commercially or can be easily produced as described in M. Tanaka, J. Chromatogr. 292 (1984) 410 or Gmelin's Handbuch der Anorganischen Chemie [Gmelin's Inorganic Chemistry Manual]
[(Springer Verlag; 8th Edition), Ferrocene Volumes 2-4, 7 and 8 (part A)~ (or analogously to the compounds described there).
The actual labeling of the desired molecule follows in the second reaction step. In this case, any selectable substance carrying NH groups, such as, e.g., a protein, a proteohormone or a polypeptide, is reacted with the radioactive metallocene carboxylic acid (derivative) of formula I, optionally by adding an activator. The reaction takes place analogously to the methods described in Analytica Chemica Acta, 1992, 264, 145-lS9.
As activators for in situ activation of the carboxylic acid groups, for example, dicyclohexylcarbodiimide (DDC), alkoxyacetylene, isobutyl chlorocarbonate, benzotriazol-l-yl-tetramethyl-uronium-tetrafluorate (TBTU), can be mentioned.
_ Other activators are described by Bodanszky et al., Principles of Peptide Synthesis, 1984.
In the case of metallocenes in which the carboxylic acid groups are present already in activated form, e.g., succinimide esters, esters, thioesters, amide or as anhydride, the addition of an activator can be dispensed with.
The second reaction step takes place quickly and in good radiochemical yields, already at temperatures in the range of 20 to 40C and is generally performed in aqueous buffer solutions, so that the requirements for a routine use in clinical practice are established.
The described "marker molecules" of formula I as well as the previously described process are suitable for radioactive labeling of any NH-active compounds, such as, for example, proteins, immunoglobulins, antibodies, especially antibodies against tumor tissue, Fab fragments, protein fragments, oligopeptides, such as, for example, endothelin or endothelin partial sequences, amino acids, neurotransmitters, proteohormones, biogenous amines, such as, for example, serotonin, histamine, y-aminobutyric acid as well as amino group-containing derivatives of steroids and estrogenic steroids, amino sugars, ergolines and other dopaminergic compounds.
The compounds labeled by using the metallocene carboxylic acids and carboxylic acid derivatives of formula I according to the mentioned process can be used directly in in vivo diagnosis and therapy, and the labeled compounds are formulated in a physiologically compatible way according to the methods usual in galenicals. This can take place, e.g., by suspending or dissolving the respective compound in a physiologically compatible suspension medium, such as water, electrolyte solutions, etc.
In in vivo use in nuclear medicine, the labeled compounds are generally dosed in amounts smaller than 10-10 mol/kg of body weight, and the exact dose can vary greatly as a function of the examined region of the body but especially also of the respectively selected method of examination.
Starting from 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 administration. Administration takes place normally intravenously, intraarterially, peritoneally or intratumorally, the intravenous administration being preferred. Generally 0.1 to 2 ml of the agent in question is administered per test.
The selection of the respective radioisotope depends on the desired diagnostic method, in which the radioactively-labeled NH-active compound is to be used.
In particular, radioisotopes Tc-99m and Ru-97 are used for the SPECT process. Relative to the short-lived Tc-99m, Ru-97 has the advantage that it can be supplied to clinics (based on its longer half-life of 2.88 days) already as a finished "marker molecule" -- for example, in the form of the metallocene acid chloride or preferably as N-succinimide ester.
Isotopes Re-186 or Re-188 are used especially in the labeling of proteins for radiation therapy.
Metallocene carboxylic acids labeled with Fe-52, Mn-52 or Ru-95 are suitable for the introduction of positron emitters into proteins.
Some of the metallocene carboxylic acids and carboxylic acid derivatives used according to the invention have not as yet been described. The invention therefore also relates to new metallocene carboxylic acids and derivatives of general formula I
~ R4 R
in which R1, R2, R3 each stand for a CO group or together mean a cyclopentadienyl radical, which optionally is substituted by R5, R~ stands for a hydrogen atom, a C,-C10 alkyl radical or a group -CO-CH3, R5, Rs, independently of one another, stand for -CO-- (CH2)~-COR6, -(CH2)n-COR6, -CH=CH-(CH2)n-COR6 or -C_C- (CH2) n-COR6, in which m stands for numbers l to 6, n stands for numbers O to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -oR7 or -SR7, with -R7 and -R8 meaning a succinimide or phthalimide radical and M stands for a Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188 or an Ir-l91.
The marker molecules of formula I are distinguished especially in that they -- do not or only insignificantly influence the biological activity of the labeled molecule, -- show a high stability in vivo and a "re-chelation" does not occur, -- can be used universally for labeling any compounds that contain NH groups and in that -- they react with compounds that contain NH groups under mild reaction conditions.
The process according to the invention is distinguished especially in that the labeling step occurs quickly and almost quantitatively under mild conditions. The process can therefore be performed easily in clinical practice, on the one hand, but also makes possible, on the other hand, the labeling of --especially thermally -- less loadable molecules, such as, e.g., proteins. Such molecules could not be radioactively labeled according to other known processes (see, e.g., W0 91/18908).
The following examples are used for a more detailed explanation of the object of the invention, without intending to be limited to this object. "Cyclopentadienylcarbonyl-technetium compounds" are designated below as "cytectrenes."
''CyClopentadienylcarbonyl-Re compounds~ are designated as "cyrhetrenes~ and ~ruthenium dicyclopentadienyl compounds" as "ruthenocenes.n Examples 1-4 describe the synthesis of various "marker molecules~ used according to the invention; Examples S-7 describe the derivatization (activation) of some "marker molecules" that contain carboxylic acid groups; Examples 8-13 describe the radioactive labeling of various NH-active compounds.
._ Metal Exchange Reaction Example ~ Tc-99m Cytectrene carboxylic acid chloride 3 mg of ferrocene carboxylic acid chloride, 0.5 mg of Mn(CO)5Br and 20 ~Ci of radioactive Tc-99m-pertechnetate, dissolved in 0.5 ml of dried methyl ethyl ketone, are added to a small glass ampoule. The ampoule is sealed off and heated for 1 hour to 150C. Then, the radioactive acid chloride is separated by thin-layer chromatography. Eluant is methylene chloride.
Rr (Cytectrene carboxylic acid chloride): 0.72 Rr (Ferrocene carboxYlic acid): 0.64 Radiochemical yield: 50%
Example 2 Tc-99m-Cytectrene carboxylic acid 1 mg of ferrocene carboxylic acid and 0.8 mg of Mn(CO)sBr are mixed with 10-20 MBq of Tc-99m-pertechnetate in 50 ~l of tetrahydrofuran, sealed in a glass ampoule under reduced pressure and heated for 1 hour to 170C. For separation of the cytectrene carboxylic acid from the radicals of unreacted ferrocene carboxylic ac d, the content of the ampoule is applied in streaks on an ICW-silicone rapid plate F 254 and chromatographed in methylene chloride/methanol (90:10). The cytectrene carboxylic acid is eluted with 2-3 ml of chloroform/methanol (1:1), and the eluate is evaporated to dryness under nitrogen atmosphere.
Rr (Cytectrene carboxylic acid): 0.30 Rr (Ferrocene carboxylic acid): 0.64 Radiochemical Yield: 91%
216097~
-Example 3 Tc-99m-Cytectrene-C0-(CH2)2-COOH
2 mg of ferrocenoylpropionic acid and 0.5 mg of Mn(C0)5Br are weighed into a small ampoule. 10 ~l of pertechnetate solution (physiological NaCl solution) with 50 MBq of Tc-99m is added to it and flushed with 50 ~l of tetrahydrofuran. After sealing off the ampoule, it is heated for 40 minutes to 150C.
The purification takes place by chromatography in cyclohexane/ether/glacial acetic acid (65:35:5). The Tc-99m-cytectrenoylpropionic acid is eluted with 1 ml of tetrahydrofuran.
Rr (Ferrocenoylpropionic acid): 0.23 Rr (Tc-99m-cytectrenoYlpropionic acid): 0.17 Radiochemical Yield: 79%
Example 4 Ru-103-ruthenocene carboxylic acid 2 mg of ferrocene carboxylic acid and a solution of 2 MBq of RU-103-C13 in 50 ~l of dioxane with 3% HCl are sealed in a glass ampoule. The contents of the ampoule are heated for 1 hour to 130C and then applied to silica gel thin-layer plates.
Chromatography in methylene chloride/methanol (88:12). After the chromatography, a yellow strip with an RF = - 64 and a non-colored, W -active strip with RF = . 56, which contains the radioactive ruthenocene carboxylic acid, are obtained. This radioactive peak is scraped off and eluted with chloroform/
methanol (1:1).
Rr (Ferrocene carboxylic acid): 0.64 Rr (Ruthenocene carboxylic acid): 0.56 Radiochemical yield: 65%
Derivatization ~Activation) Example 5 Tc-ssm-cytectrene carboxylic acid-N-succinimide ester The cytectrene carboxylic acid obtained from Example 2 is taken up in 200 ~1 of tetrahydrofuran and reacted at room temperature with 0.25 mg of N-hydroxysuccinimide as well as 0.5 mg of dicyclohexylcarboxylic acid diimide for 30 minutes with stirring. Purification takes place by thin-layer chromatography in methylene chloride/methanol (98:2).
Rr (N-Succinimide ester): 0.57 Radiochemical Yield: 92%
Example 6 Tc-99m-Cytectrenoylpropionic acid-N-succinimide ester 200 ~1 of eluate of the Tc-99m-cytectrenoylpropionic acid of Example 3, 4 mg of N-hydroxysuccinimide and 8 mg of dicyclohexylcarboxylic acid diimide are allowed to stand at room temperature with repeated shaking. After 2 hours, the N-succinimide ester is obtained by chromatography in 79% yield.
The purification takes place by thin-layer chromatography in methylene chloride/methanol (95:5).
Rr (N-Succinimide ester): 0.65 Radiochemical yield: 79%
Example 7 Ru-103-Ruthenocene carboxylic acia-N-suc¢inimide e~ter The eluate of the radioactive ruthenocene carboxylic acid obtained in Example 4 is evaporated to dryness, mixed with 5 mg of N-hydroxysuccinimide and 10 mg of N,N-dicyclohexylcarbodiimide in 300 ~l of tetrahydrofuran. Then, it is stirred for 1 hour at room temperature. The ruthenocene carboxylic acid-N-succinimide ester is obtained after thin-layer chromatography in methylene chloride/methanol (88:12) in 95% yield.
Rr (Ruthenocene carboxYlic acid): 0.49 Rr fRuthenocene carboxYlic acid-N-succinimide ester): 0.82 Radiochemical yield: 95~ -Labeling Example 8 Tc-99m-Cytectrenoyl-glycine The solution of cytectrene carboxylic acid-N-succinimide ester obtained from Example 5 is concentrated by evaporation under nitrogen atmosphere to 10 ~l and mixed with 3 ~mol of glycine -- dissolved in 100 ~l of borate buffer [0.1 molar, pH
8.5]. After 1 hour of stirring 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)-Rr (Tc-99m-Cytectrenoyl-glycine): 0.30 Radiochemical yield: 82%
17 216097~
Example 9 ~c-gsm-cytectrenoyl-glycine-ethyl ester 10.93 MBq of Tc-99m-cytectrene carboxylic acid produced according to Example 2 is dissolved in 2 ml of tetrahydrofuran.
0.2 ml is removed from this solution and mixed with 2 mg of benzotriazol-l-yl-tetramethyl-uronium-tetrafluorate and 1 ml of diisopropylethylamine. After S minutes of shaking at room temperature, this solution is added to 1 mg of glycine ethyl ester x HCl in 0.1 ml of tetrahydrofuran and 2 ~1 of diisopropylamine. The reaction mixture is stirred for 30 minutes. The thin-layer chromatography takes place in chloroform/acetone/formic acid (80:2, 5:2, S).
Rr (Ferrocene carboxylic acid): 0.39 Rr (Ferrocenoyl-glycine ethyl ester): 0.58 Rr (Tc-99m-Cytectrenoyl-glycine ethYl ester): 0.62 Radiochemical yield of Tc-99m-cytectrenoYl-glycine ethYl ester: 98%
Example 10 Tc-99m-Cytectrenoyl-N-glucosamine 0.76 mg (2 ~mol) of HBTU, 1 ~1 of diisopropylamine and 100 ~1 of methyl pyrrolidone are added to 9 MBq of Tc-99m-labeled cytectrene carboxylic acid produced according to Example 2. The reaction mix~ure is stirred for 30 minutes at room temperature and then applied to 20 cm-wide TLC plates. In the mobile solvent chloroform/e~hanol (70:30) at an RF of 0.40, a radioactivity maximum of t~e desired glucosamine derivative is obtained. After scraping off this RF area, the radioactive substance is eluted 18 216~979 -with methanol. Re-chromatography in acetone/ethanol/ammonia (75:20:5) shows the radiochemical purity of this compound.
Rr (CYtectrenoyl-N-glucosamine): 0.40 Radiochemical Yield: 80%
Example 11 Ru-103-Ruthenocenoyl-glycine-ethyl ester After removal of the solvent, 1 mg of glycine ethyl ester and 100 ~l of borate buffer (pH 9) are added to 1 MBg of the ruthenocene carboxylic acid-N-succinimide ester of Example 7.
After 1 hour, 69% is obtained, and after 2 hours, 91% of the desired ruthenocenoyl-glycine ethyl ester is obtained. The purification takes place by thin-layer chromatography in chloroform/acetone/formic acid (80:2, 5:2, 5).
Rr (RuthenocenoYl-glYcine-ethYl ester): 0.43 Rr (Ruthenocene carboxylic acid-N-hydroxYsuccinimide ester):
0.56 Radiochemical yield: 91%
~xample 12 Ru-103-Ruthenocenoyl-heptapeptide 1 mg of endothelin derivative [heptapeptide; Gly-His-Leu-Asp-Ile-Ile-Trp-COOH] with 4.8 KBq of Ru-103-ruthenocene carboxylic acid-N-succinimide ester (produced according to Example 7) is stirred in a small glass in 100 ~1 of borate buffer (pH 9) at room temperature. After 90 minutes, the batch is chromatographed in the mobile solvent of chloroform/methanol/
ammonia (50:4S:10).
Radiochemical Yield: 62%
19 216097~
Example 13 Tc-99m-Cytectrenoyl-heptapeptide 19 MBq of cytectrene carboxylic acid-N-succinimide ester (produced according to Example 5) in 0.1 ml of THF is mixed with 1 mg of endothelin derivative [heptapeptide; Gly-His-Leu-Asp-Ile-Ile-Trp-CoOH] dissolved in 0.2 ml of borate buffer (pH 9). After 180 minutes of stirring, the batch is chromatographed in the mobile solvent ethyl acetate/MeOH/NH3 (50:40:10).
Rr Cytectrene carboxYlic acid succinimide ester: 0.69 Rr (Cytectrenoyl-heptapeptide): 0.33 Radiochemical yield: 53.6%
Claims (10)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Use of metallocene carboxylic acids and their derivatives of radioactive metal isotopes of general formula I
(I) in which R1, R2, R3 each stand for a CO group or together mean a cyclopentadienyl radical, which optionally is substituted by R5, R4 stands for a hydrogen atom, a C1-C10 alkyl radical or a group -CO-CH3, R5, R5', independently of one another, stand for -CO-(CH2)m-COR6, -(CH2)n-COR6, -CH=CH-(CH2)n-COR6 or -CC-(CH2)n-COR6, in which m stands for numbers 1 to 6, n stands for numbers 0 to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -OR7 or -SR7, with R7 meaning hydrogen, a C1-C10 alkyl radical, or radical, and R8 in the meaning indicated for R7 with the exception of hydrogen and a C1-C10 alkyl radical and M means a radioactive metal isotope of an element of atomic numbers 21-29, 31, 42-44, 49 or 57-83 for radioactive labeling of compounds that contain NH groups.
(I) in which R1, R2, R3 each stand for a CO group or together mean a cyclopentadienyl radical, which optionally is substituted by R5, R4 stands for a hydrogen atom, a C1-C10 alkyl radical or a group -CO-CH3, R5, R5', independently of one another, stand for -CO-(CH2)m-COR6, -(CH2)n-COR6, -CH=CH-(CH2)n-COR6 or -CC-(CH2)n-COR6, in which m stands for numbers 1 to 6, n stands for numbers 0 to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -OR7 or -SR7, with R7 meaning hydrogen, a C1-C10 alkyl radical, or radical, and R8 in the meaning indicated for R7 with the exception of hydrogen and a C1-C10 alkyl radical and M means a radioactive metal isotope of an element of atomic numbers 21-29, 31, 42-44, 49 or 57-83 for radioactive labeling of compounds that contain NH groups.
2. Use of the compounds according to claim 1 for labeling proteins, immunoglobulins, antibodies, Fab fragments, protein fragments, oligopeptides, amino acids, histamine, .gamma.-aminobutyric acid, neurotransmitters, proteohormones, biogenous amines, NH-group-containing derivatives of steroids and estrogenic 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 general formula I
(I) in which R1, R2, R3 each stand for a CO group or together mean a cyclopentadienyl radical, which optionally is substituted by R5', R4 stands for a hydrogen atom, a C1-C10 alkyl radical or a group -CO-CH3, R5, R5', independently of one another, stand for -CO-(CH2)m-COR6, -(CH2)n-COR6, -CH=CH(CH2)n-COR6 or -CC-(CH2)n-COR6, in which m stands for numbers 1 to 6, n stands for numbers 0 to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -OR7 or -SR7, with -R7 and -R8 meaning a succinimide or phthalimide radical and M stands for a Cr-51, Mn-52, Ru-97, Ru-103, Re-186, Re-188 or an Ir-191.
(I) in which R1, R2, R3 each stand for a CO group or together mean a cyclopentadienyl radical, which optionally is substituted by R5', R4 stands for a hydrogen atom, a C1-C10 alkyl radical or a group -CO-CH3, R5, R5', independently of one another, stand for -CO-(CH2)m-COR6, -(CH2)n-COR6, -CH=CH(CH2)n-COR6 or -CC-(CH2)n-COR6, in which m stands for numbers 1 to 6, n stands for numbers 0 to 6, R6 stands for chlorine, a group -NH-NH2, a radical -R8, -OR7 or -SR7, with -R7 and -R8 meaning a succinimide or phthalimide radical 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-ruthenocene carboxylic acid chloride.
6. Metallocene carboxylic acid derivative according to claim 4, namely Ru-97-ruthenocene carboxylic acid-N-succinimide ester.
7. Process for the production of radioactively-labeled NH-active substances, characterized in that a compound of formula I
(I) in which R1, R2, R3, R4 and R5 have the indicated meaning, is reacted optionally with the addition of an activator, in a suitable solvent with an NH-active substance at 20°C - 40°C.
(I) in which R1, R2, R3, R4 and R5 have the indicated meaning, is reacted optionally with the addition of an activator, in a suitable solvent with an NH-active substance at 20°C - 40°C.
8. Process according to claim 7, in which R5 contains a -COCl radical, a succinimide radical or an N-hydroxysuccinimide radical.
9. Process according to claim 7, wherein Ru-97-ruthenocene carboxylic acid-N-succinimide ester, Tc-99m-cytectrene carboxylic acid-N-succinimide ester or a radioactive cyrhetrene carboxylic acid-N-succinimide ester is used as a radioactive metallocene carboxylic acid derivative of formula I.
10. Process according to claim 7, wherein dicyclohexylcarbodiimide or benzotriazol-1-yl-tetramethyl-uronium-tetrafluorate is used as an in situ activator.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19934313670 DE4313670A1 (en) | 1993-04-22 | 1993-04-22 | Radioactive metallocene carboxylic acids and their homologues for protein labeling |
| DEP4313670.2 | 1993-04-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2160979A1 true CA2160979A1 (en) | 1994-10-27 |
Family
ID=6486432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2160979 Abandoned CA2160979A1 (en) | 1993-04-22 | 1994-04-20 | Use of cyclopentadienylcarbonyl transition metal carboxylic acids and their derivatives for labeling proteins |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0696207A1 (en) |
| JP (1) | JPH08508985A (en) |
| CA (1) | CA2160979A1 (en) |
| DE (1) | DE4313670A1 (en) |
| WO (1) | WO1994023757A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006026855A1 (en) * | 2004-09-07 | 2006-03-16 | Triumf, Operating As A Joint Venture By The Governors Of The University Of Alberta, The University Of British Columbia, Carleton | Synthesis of radiolabeled sugar metal complexes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE59104905D1 (en) * | 1990-06-01 | 1995-04-13 | Diagnostikforschung Inst | 99m-Tc-CYCLOPENTADIENYLCARBONYL COMPLEXES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN DIAGNOSTICS. |
-
1993
- 1993-04-22 DE DE19934313670 patent/DE4313670A1/en not_active Withdrawn
-
1994
- 1994-04-20 WO PCT/EP1994/001250 patent/WO1994023757A1/en not_active Application Discontinuation
- 1994-04-20 CA CA 2160979 patent/CA2160979A1/en not_active Abandoned
- 1994-04-20 JP JP6522787A patent/JPH08508985A/en active Pending
- 1994-04-20 EP EP94915087A patent/EP0696207A1/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006026855A1 (en) * | 2004-09-07 | 2006-03-16 | Triumf, Operating As A Joint Venture By The Governors Of The University Of Alberta, The University Of British Columbia, Carleton | Synthesis of radiolabeled sugar metal complexes |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0696207A1 (en) | 1996-02-14 |
| JPH08508985A (en) | 1996-09-24 |
| WO1994023757A1 (en) | 1994-10-27 |
| DE4313670A1 (en) | 1994-10-27 |
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