IE920761A1

IE920761A1 - Chelates, their metal complexes as well as their use in¹diagnosis and treatment

Info

Publication number: IE920761A1
Application number: IE076192A
Authority: IE
Inventors: Dr Reinhard Neumeier; Dr Wolfgang Kramp; Dr Helmut R Macke; Dr Gerhard Rohlfs
Original assignee: Diagnostikforschung Inst
Priority date: 1991-03-07
Filing date: 1992-03-09
Publication date: 1992-09-09
Also published as: PT100200A; ZA921716B; AU1214892A; JPH06100519A; EP0502595A2; CA2062454A1; EP0502595A3; DE4107571A1

Abstract

The invention relates to compounds of the general formula I in which A comprises a functional group which is suitable for coupling to compounds which undergo selective accumulation, and R<1>, R<2>, R<3>, R<4> and R<5> may have a variety of meanings. B is a group which is suitable for the coordinative bonding of metal ions. The compounds are used for the complexation of radioactive metal ions, especially rhenium and technetium isotopes, and are used in medical diagnosis and therapy.

Description

Radioactive metal ions, mostly bound to a complexing agent, have for some time been used for in vivo diagnosis. Among them, technetium-99m (Tc-99m), because of its almost ideal physical properties for this purpose — good absorption of radiation in corresponding detection devices (gamma camera, SPECT devices) relative to a low absorption in the human organism and easy availability with a molybdenum/technetium generator — is the radionuclide most often used in clinical nuclear medicine. Its short half-life of 6.02 hours guarantees an only slight exposure of the patient to gamma radiation, particularly also the daughter product technetium-99 has only a negligible residual radiation. But a drawback of the technetium is its complicated and still not completely known complex chemistry. Technetium can be present in a number of oxidation stages (+7 to -1), which can greatly change the pharmacological properties by changing the charge of a complex. It is therefore necessary to synthesize complexes which bind the technetium in a defined oxidation stage and to prevent redox reactions, which could lead to a redistribution of the pharmaceutical agent. A number of such Tc-99m complexing agents are already known and are clinically used. In the case of neutral complexes, systems, in which the Tc-99m is bound between 2-4 nitrogen atoms and 0-2 sulfur atoms (N2S2, NgS and propylenamine oxime complexes), are often involved. But often the insufficient stability of these Tc-99m complexes is a significant drawback (Hung, J. C. et al.; J. Nucl. Med. 29: 1568, 1988). In clinical use, therefore, e.g., HMPAO (hexamethyl-propylenamine oxime) has to be administered shortly after its labeling with pertechnetate, so that the portion of decomposition products, which reduce the diagnostic informative value, is not too high.

A coupling of these chelates or chelating agents to other substances selectively accumulating in foci of disease is not possible. Therefore, most of said complexes are distributed according to the blood circulation and/or metabolic activity of an organ (e.g., Europ. Patent Appl. 0 194 843), so that, e.g., necrotic or ischemic regions can be represented after infarction or stroke in a scintigram.

But for a successful diagnosis of tumors, neurological diseases or diseases of the cardiovascular system, substances are more promising which can produce molecular changes of diseased tissue, by being bound specifically to these diseased tissues or being infiltrated in their metabolism. The findings of the biological and biochemical basic research allow the selection of a number of substances, which selectively accumulate in foci of disease: various tumors develop increased or reduced surface concentrations on receptors, e.g., for growth factors or steroid hormones (Sledge, G. W.; Adv. Cancer Res. 38: 61-75 [1983]).

Also, in neurological diseases, a change of the concentration of receptors for neutrotransmitters results in specific areas of the brain (Frost, J. J.; Trends Pharmacol. Sci. 7: 490-496 [1987]). Further, diseased, damaged or cells transformed into tumor cells often show great changes of their metabolism and an oxygen deficiency inside the tumor. The use of such physiological characteristics can be used in in vivo diagnosis, by, e.g., hormones, neurotransmitters or certain metabolic products such as fatty acids, saccharides, peptides or amino acids being coupled to chelating agents for Tc-99m. Also, substances such as misonidazole (a radiosensitizer) or other compounds reacting to radicals in the absence of oxygen can be used for specific accumulation of radioactive isotopes and thus graphic representation of tumors or ischemic regions. Finally, also the coupling to monoclonal antibodies is possible, which because of their high specificity have become a promising instrument in the diagnosis of tumors.

For the production of diagnostic agents according to the described principle, it is necessary that chelating agents for radioactive metal ions, in particular Tc-99m, can be coupled to substances selectively accumulating in diseased tissues.

Since the isotopes of rhenium (Re-188 and Re-186) have chemical properties similar to Tc-99m, the chelating agents can also be used to complex these isotopes. Said Re-isotopes are βradiators. Thus, the selectively accumulating substances complexed with rhenium instead of with technetium are also usable in the treatment of tumors.

The previously known attempts to couple chelating agents to selectively accumulating substances in many cases can be considered as unsatisfactory. If the functional groups of the ' complexing agent are used to bind the chelating agent to such a molecule, a weakening of the complex stability often results, i.e., a diagnostically intolerable portion of the isotope is released from the conjugate. (Brechbiel, M. W. et al., Inorg. Chem. 25: 2772 [1986]). It is therefore necessary to produce bifunctional complexing agents, i.e. complexing agents which carry both functional groups for coordinative bonding of the desired metal ion and a (another) functional group for bonding the selectively accumulating molecule. Such bifunctional ligands make possible a specific, chemically defined bonding of technetium to the most varied biological materials, also then when a so-called prelabeling is performed. Since according to this method, first the labeling with Tc-99m and the isolation of the complexes is performed and this complex is linked only in a second step with a selectively accumulating molecule, the labeled compounds are obtained with a high degree of purity.

Some chelating agents coupled to monoclonal antibodies (e.g., Europ. Patent App. 0 247 866 and 0 188 256) or fatty acids (Europ. Patent Appl. 0 200 492) were described. But as chelating agents, the already mentioned N2S2 systems are used, which are not very suitable because of their low stability. The somewhat more stable N3S chelates showed, coupled to monoclonal antibodies, not so great a loss of Tc-99m from the conjugates (J. Lister-James; J. Nucl. Med. 30: 793 and Europ. Patent Appl. 0 284 071) .

Since both the selectively accumulating substances in their properties and the mechanisms, according to which they are accumulated, are very different, it is further necessary to be able to vary the couplable chelating agents and match the physiological requirements of the coupling participant with respect to lipophilia and hydrophilia, membrane permeability or impermeability, etc.

For these reasons, there exists an urgent need for stable complex compounds, which are coupled or able to couple to various selectively accumulating compounds.

The object of the invention is thus to make available stable chelating agents, which contain a functional group for coupling to a selectively accumulating compound or a selectively accumulating compound coupled with the help of this functional group.

According to the invention, this object is achieved by the compounds of general formula I in which R1 stands for hydrogen or a C1-6 alkyl radical optionally substituted with one or two hydroxyl groups, R2 stands for a 0χ_6 alkylene radical optionally substituted with a hydroxyl group, R3 stands for a hydrogen atom, a alkyl radical, a carboxymethyl radical or a (Cy“g alkoxycarbonyl)methyl radical, R4 stands for a hydrogen atom, a alkyl radical optionally substituted with a hydroxyl group or for the meaning indicated under Rx, R5 stands for a hydrogen atom or a alkyl radical optionally substituted with a hydroxyl group, B stands for a pyrrolyl radical, a substituted phenyl radical of formula II or a substituted pyridine radical of formula III in which Z means a hydroxyl group, amino group or a mercapto group and Y means a hydrogen atom, a carboxy radical or a sulfonyl radical or B stands for a nitrosomethyl radical of formula IV NOH S' !/ 7-c Rx (IV) • - x 4 in which R means a C-j_g alkyl radical or, together with P , is a trimethylene or tetramethylene grouo to form a 5- or 6-membered ring and A stands for an amino radical, a mercapto radical, a carboxy radical, a C2_6 alkinyl or C2_6 alkenyl radical, an oxiranyl radical, a fluorinated phenoxycarbonyl radical, a succinimidoxycarbonyl radical optionally substituted with a sodium sulfate radical, an aminophenyl radical or isothiocyanatophenyl radical, and the phenyl group optionally can be substituted in addition by a carboxy radical, a chlorosulfonyl radical or a sulfonic acid radical or contains a compound T selectively accumulating in lesions or specific tissues — optionally bound by a bifunctional linker radical L with the help of said functional group and T stands for monoclonal antibodies or their fragments, hormones, enzymes, ligands for cell membrane receptors, neurotransmitters, lipids, steroids, saccharides, amino acids and oligopeptides, biotin, as well as radiosensitizers, such as, e.g., misonidazole, NOH II and in the case of B = -C, pyrrolyl radical or I Rx Y = H, R2 is to stand for a hydroxylated Cj_6 alkylene chain or A is to stand for a substituted aminophenyl or isothiocyanatophenyl radical, as well as their complexes with radioactive metal ions — suitable for diagnosis and treatment of tumors — as well as their salts with inorganic and organic acids or bases.

According to the invention, those compounds according to claim 1 are preferred in which R4 and R5 mean hydrogen atoms or methyl radicals, as well as those compounds according to claim 1, in which A stands for an amino radical, a mercapto radical, a carboxy radical, a C2_6 alkinyl or C2_6 alkenyl radical, an oxiranyl radical, a fluorinated phenoxycarbonyl radical, a succinimidoxycarbonyl radical optionally substituted with a sodium sulfate radical, an aminophenyl or isothiocyanatophenyl radical, and the phenyl group can be substituted by another carboxyl or sulfonic acid radical, and the selectively accumulating compound T optionally contained in A stands for monoclonal antibodies, their fragments, biotin or misonidazole.

Surprisingly, many of the chelates synthesized and labeled with Tc-99m showed a higher stability than the comparable N2S2, N3S and propylenamine oxime chelates. Thus, no decomposition products were able to be observed after 5 hours, e.g., in a substance according to the invention (example 2), which was coupled by an aminophenyl radical to biotin, while this was the case in the comparable HMPAO known in the literature (Hung, J. C. et al.; J. Nucl. Med. 29: 1568, 1988). Also, by competitive tests, it was possible to be determined that the Tc-99m chelates described in this invention complex better than the comparable N2S2, N3S and propylenamine oxime chelates. The chelates described in the invention are thus definitely better suitable for diagnostic and therapeutic purposes than the previously known chelates.

The production of the compounds according to claim 1 takes place, by a) a 1,3-propanediamine of general formula V in which R1 and R2 have the above-mentioned meanings, and A' means A or a radical able to be converted to A being reacted with a compound of general formula VI B - R4C = Ο (VI) in which R4 and B have the above-mentioned meanings, in a polar solvent, preferably ethanol, or by using a water separator in a nonpolar solvent, preferably benzene, at temperatures of 25-180°C within 6 hours to 3 days, the imino function being reduced in a way known in the art, preferably with sodium borohydride in a polar solvent, preferably a methanol/water mixture, at temperatures of 25-100°C within 0.5 to 24 hours, preferably 2 hours, or b) by a propanediamine of general formula V R\ R2A’ H2N NH2 (V) (V) in which R1, R2 and A' have the above-mentioned meanings, being reacted with a compound of general formula VII B - CO-X (VII), in which B has the above-mentioned meaning, functional groups contained in B optionally are present in protected form and X stands for a halogen atom, preferably for a chlorine atom, or c) by a propanediamine of general formula V (V) in which R1, R2 and A' have the above-mentioned meanings, being reacted with a compound of general formula VIII R4 / 5 B—C— i X (VIII) in which R4, R5, B and X have the above-mentioned meanings and functional groups contained in B optionally are present in protected form, or d) ' substituted malonic acid halides, preferably malonic acid chlorides of general formula IX R\ R2A' XOC COX (IX) in which R3, R2, k' and X have the above-mentioned meanings and functional groups contained in B optionally are present in protected form, being reacted with an amine of general formula X R4 B—C——R5 NHR3 (X) in which R4, R5 and B have the above-mentioned meanings, R3' stands for a hydrogen atom or for a alkyl radical and functional groups contained in B optionally are present in protected form, in an aprotic solvent, preferably dichloromethane, at temperatures of 0-180°C, preferably at room temperature, within 2 to 24 hours, preferably 4 hours, optionally by adding suitable bases, e.g., triethylamine, the amide function obtained according to process d) being reduced in a way known in the art, preferably with borane in THF or with lithium aluminum hydride in an aprotic solvent, preferably diethyl ether, at temperatures of 25-150°C within 0.5 to 24 hours, preferably 8 hours to the corresponding amino function, the amino groups, in the case that R^ stands for a hydrogen atom, optionally being alkylated in a way known in the art with an alkylating agent introducing R3 and present protecting groups being cleaved and in the thus obtained compounds, group A' optionally converted to A — optionally after protecting the free amino groups and functional groups Z with protective ions, e.g., as a Cu complex — being generated and then optionally the thus obtained compounds being coupled by these functional groups to selectively accumulating compounds T and substituents B optionally being complexed with the radioactive isotope desired in each case, and protective ions optionally present in the product in advance being removed according to methods known in the literature and the sequence of the steps of complexing with radioactive isotopes and coupling to T being able to be interchanged.

As hydroxy protecting groups, e.g., the benzyl, 4methoxybenzyl, 4-nitrobenzyl, trityl, diphenylmethyl, trimethylsilyl, dimethyl-t-butylsilyl and diphenyl-t-butylsilyl groups are suitable. In the case of polyols, the hydroxy groups can also be protected in the form of ketals with, e.g., acetone, acetaldehyde, cyclohexanone or benzaldehyde. Further, the hydroxy groups can also be present, e.g., as THP-ether, «alkoxyethyl ether, MEM ether or as esters with aromatic or aliphatic carboxylic acids, such as, e.g., acetic acid or benzoic acid. The hydroxy protecting groups can be released according to methods in the literature known to one skilled in the art, e.g., by hydrogenolysis, reductive cleavage with lithium/ammonia, acid treatment of the ethers and ketals or alkali treatment of the esters (see, e.g., Protective Groups in Organic Synthesis, T.

W. Greene, John Wiley and Sons 1981).

As amino protecting groups, e.g., trifluoroacetyl, tbutoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzoxycarbonyl and acetyl groups are suitable. The amino protecting groups can be cleaved according to methods known in the literature, e.g., by basic or acid hydrolysis, reductive cleavage with zinc in acetic acid or hydrogenolysis.

As mercapto protecting groups, alkyl radicals or benzylthioether are suitable. The cleavage of the mercapto protecting groups takes place selectively with alkali alkylthiolates, alkali alcoholates or alkali metals, preferably with sodium methylthiolate in a polar solvent, preferably in HMPT, DMF or dimethylacetamide or by reductive cleavage with sodium/ammonia.

The functional group in radical A is suitable for producing a stable compound to proteins or other selectively accumulating molecules. By the corresponding selection of the functional group in radical A, the coupling is possible under gentle reaction conditions, which do not influence the biological function and/or selectivity.

The coupling to the desired compounds also takes place according to methods known in the art (e.g., Fritzberg et al.; J. Nucl. Med.: 26, 7 [1987]), for example, by reaction of the corresponding functional groups in radical A with nucleophilic groups of the selectively accumulating molecule or, if a nucleophile is involved in the corresponding functional group in radical A itself, with activated groups of the selectively accumulating molecule.

The functional group in radical A represents every substituent which on the one hand allows a coupling to a selectively accumulating molecule under mild conditions (e.g., by acylation or amidation) as well as every activated group, which can react with nucleophilic groups of proteins, antibodies, hormones or other biomolecules, such as the amino, phenol, sulfhydryl, formyl or imidazole group. By activated group is to be understood a function which is capable of reacting with the formation of a conjugate with a nucleophilic substituent of a selectively accumulating molecule or of the complex ligand itself in aqueous solution within a suitably short time, under reaction conditions which entail neither denaturation nor loss of the biological activity. Examples in this respect are imide esters, alkylimide esters, amidoalkylimide esters, succinimide esters, acylsuccinimides, phenol esters, substituted phenol esters, tetrafluorophenol esters, anhydrides, and Michael acceptors. The functional group in radical A is preferably an activated ester (such as phenol or imide ester), a mercaptan or isothiocyanate, in particular for the coupling with nucleophilic groups of amino acids or an aliphatic or aromatic primary amine for the coupling to carbohydrate radicals of proteins.

If a nucleophile is involved in the functional group in radical A itself, it can react with activated groups of a selectively accumulating molecule, and groups of the molecule reacted with so-called crosslinking reagents are also enclosed, I such as homobifunctional imido esters, homobifunctional Nhydroxysuccinimide esters (NHS) and heterobifunctional '’crosslinkers, ” which contain various functional groups, such as NHS esters, pyridyl disulfides and activated halogens, such as eketo halides. Such crosslinkers are commercially available.

As coupling participants (= compound T), i.a., various biomolecules are provided: ligands, which bind to specific receptors and thus can detect a tissue changed in its receptor density; they include, i.a., peptide and steroid hormones and neurotransmitters. With ligands for steroid hormone receptors, the possibility of an improved diagnosis of breast and prostate carcinomas was demonstrated (S. J. Brandes & J. A.

Katzenellenbogen, Nucl. Med. Biol. 15: 53, 1988). Often, ligands labeled with positron-emitting isotopes for neuroreceptors were able to be used for diagnosis of various brain diseases (J. J. Forst, Trends in Pharmacol. Sci. 7: 490, 1987). Other biomolecules are metabolites able to infiltrate the metabolism of the cells, which make a changed metabolism recognizable; they include, for example, fatty acids, saccharides, peptides and amino acids. Fatty acids coupled to the more unstable N2S2 chelating agents were described in EPA 0 200 492. Other metabolism products such as saccharides (dexoxyglucose), lactate, pyruvate and amino acids (leucine, methylmethionine, glycine) were used with the help of the PET technique for graphic representation of changed metabolic processes (R. Weinreich, Swiss Med. 8, 10, 1986). Also, nonbiological substances such as misonidazole and its derivatives, which in tissues or tissue parts with reduced oxygen concentration bind irreversibly to cell components, can be used for specific accumulation of radioactive isotopes and thus graphic representation of tumors or ischemic regions (Μ. E. Shelton, J. Nucl. Med. 30: 351, 1989). Finally, the coupling of the bifunctional chelating agents to monoclonal antibodies or their fragments is also possible. The compounds according to the invention containing biotin make possible the bonding of the radioactive conjugates to substances containing avidin or streptavidin. This can be used to accumulate antibodystreptavidin conjugates on the tumor and only later to administer the radioactive biotin-containing components, which results in a reduced exposure of the patient to radioactive radiation (D. J. Hnatowich et al., J. Nucl. Med. 28: 1294, 1987). By complexing the conjugates with Tc-99m or rhenium isotopes, a diagnosis and treatment of tumors can be made possible.

It is unimportant whether a labeling of the chelating agents with Tc-99m or a rhenium isotope is performed before or after the coupling to the selectively accumulating molecule. But for a coupling to the selectively accumulating molecule after a complexing, it is a prerequisite that the reaction of the radioactive complex with the accumulating compound occurs quickly, under gentle conditions and almost quantitatively, and no subsequent purification is necessary.

The production of the pharmaceutical agents according to the invention takes place in a way known in the art, by the complexing agents according to the invention being dissolved while adding a reducing agent, preferably tin (II) salts such as tin chloride or tin tartrate — and optionally by adding the additives usual in galenicals — in aqueous medium and then being sterilized by filtration.

Suitable additives are, for example, physiologically harmless buffers (e.g., tromethamine), small additions of electrolytes (e.g., sodium chloride), stabilizers (e.g., gluconate, phosphates or phosphonates). The pharmaceutical agent according to the invention is present in the form of a solution or in freeze-dried form and is mixed shortly before the administration with a solution of Tc-99m pertechnetate, eluted from commercially available generators, or a perrhenate solution.

In the nuclear medical in vivo use, the agents according to the invention are provided in amounts of l*10~5 to 5* 104 nmol/kg of body weight, preferably in amounts between 1·1Ο-3 and 5* 102 nmol/kg of body weight. Starting from an average body weight of 70 kg, the amount of radioactivity for diagnostic uses is between 0.05 and 50 mCi, preferably 5 to 30 mCi per administration. For therapeutic uses, between 5 and 500 mCi, preferably 10-350 mCi is administered. The administration is normally performed by intravenous, intraarterial, peritoneal or intratumoral injection of 0.1 to 2 ml of a solution of the agent according to the invention. The intravenous administration is preferred.

The following examples are used to explain the object of the invention in more detail. i Example 1: 2-Methyl-2-(4-nitrobenzyl)-malonic acid dimethyl ester ill 11.5 g (0.5 mol) of sodium under a nitrogen stream is added in a dry three-necked flask holding 1000 ml with a reflux condenser and drying tube as well as a dropping funnel with pressure compensation. Then, 350 ml of methanol is carefully instilled and stirred until the sodium is completely dissolved.

In the still warm sodium raethanolate solution, the methanolic solution of 87 g (0.5 mol) of methylmalonic acid diethyl ester is instilled very slowly. After completion of the addition, it is stirred for another 30 minutes and then the 4-nitrobenzyl bromide is added in portions with the help of a powder funnel. After everything has dissolved, it is first refluxed for 2 hours and then stirred overnight at room temperature. The solvent is drawn off on a rotary evaporator, the residue is mixed with water and extracted several times with ethyl acetate (250 ml each time).

The combined organic extracts are washed with saturated common salt solution and dried on sodium sulfate. After removal of the solvent, pale yellow crystals remain. The recrystallization takes place from dimethylformamide (DMF)/water.

Melting point: 94.5 - 95.0°C Yield: 74% 1H-NMR data in DMSO/TMS 1.4 ppm (s,3H,Me); 3.5 ppm (s,2H,CH2Ar); 3.8 ppm (s,6H,MeOOC); 7.2 ppm (d,2H,ArH); 8.2 ppm (d,2H,ArH) ,Ε 920761 2-Methyl-2-(4-nitrobenzyl)-malonic acid diamide Γ21 .0 g of ester [1] is weighed in in a 500 ml round-bottom flask and dissolved in 200 ml of methanol. In this methanolic solution, ammonia is introduced to saturation, then the catalytic amount of sodium is added and after completion of the gas generation, the flask is closed with a suction piece with balloon and allowed to stand for at least 1 week at room temperature. After several days, a white precipitate precipitates. After all the feedstock is reacted (DC control) , the precipitated precipitate is suctioned off and the remaining solution is cooled in the freezer to -20°C and again suctioned off. The mother liquor is further concentrated by evaporation on a rotary evaporator and the precipitated precipitate is again suctioned off. The recrystallization of the combined crystal fractions takes place from acetonitrile. The white crystalline product is dried in a vacuum dessicator.

Melting point: 179°C Yield: 91% 1H-NMR data in DMSO/TMS 1.2 ppm (s,3H,Me); 3.3 ppm (s,2H,CH2Ar); 7.1 ppm (s(br),4H,CONH2); 7.3 ppm (d,2H,ArH); 8.1 ppm (d,2H,ArH) 2-Methvl-2-(4-nitrobenzvl)-1,3-propanediamine f31 Diamide [2] (5.0 g) is suspended with exclusion of atmospheric moisture in 25 ml of abs. tetrahydrofuran in a 250 ml two-necked flask with a reflux condenser and drying tube as well as a septum. The flask is cooled down in an ice bath and then the borane solution in THF is added with the help of a 20 or 50ml one-way syringe by the septum. After the necessary amount of the borane solution (80 ml of a 1 M solution in THF) was added, it was allowed to heat to room temperature. After 30 minutes, the mixture is refluxed for 4 hours. After cooling to room temperature, the excess of borane is hydrolyzed carefully with water (a total of 16 ml of water). After completion of the gas generation, the reaction mixture is converted quantitatively in a 500 ml round-bottom flask and the solvent is drawn off carefully on a rotary evaporator, a white precipitate remains. This precipitate is mixed with 125 ml of 6 N HCl and refluxed for 3 hours. Then, the hydrochloric acid is drawn off slowly on a rotary evaporator. The residue is taken up in the minimum amount of water (about 50-70 ml) and added to a prepared ion exchanger column (strongly basic) and the pure amine is eluted with distilled water. The free diamine is collected (pH control) and the combined fractions are concentrated by evaporation on a rotary evaporator. A pale yellow oil remains as residue.

Melting point (hydrochloride): 270°C Yield: 80% 1H-NMR data in DMSO/TMS 1.0 ppm (s,3H,Me); 2.9 ppm (m,4H,CH2NH2); 3.0 ppm (s,2H,CH2Ar); 7.5 ppm (d,2H,ArH); 8.2 ppm (d,2H,ArH); 8.5 ppm (s(br),4H,NH2) 2-Methyl-2-(4/-nitrobenzvl)-N,N/-propvlene-bis-r(5sulfo)salicylidenaminel Γ41 A solution of 5.6 g of diamine [3] in 100 ml of 50% ethanol is adjusted with acetic acid to pH 5-6. 8.6 g of 5sulfosalicylaldehyde and 1.9 g of sodium cyanoborohydride are added to this solution and stirred overnight. After cooling to 0°C, it is adjusted with semiconcentrated hydrochloric acid to pH 2 and concentrated by evaporation. The residue is neutralized with saturated potassium carbonate solution and then freezedried. The residue is extracted with DMF/MeOH. After removal of the solvent, a crystalline residue remains which is recrystallized from acetonitrile/water.

Yield: 56% 1H-NMR data in DMSO/TMS 0.9 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 2.8 ppm (s,2H,CH2Ar); 4.1 ppm (m,4H,ArCH2NH); 7.0-7.8 ppm (m,8H,ArH); 8.2 ppm (d,2H,ArH) 2-Methvl-2-(4z-aminobenzyl)-N,N'-propylene-bis-r(5sulfolsalicylidenaminel Γ51 mg of 10% Pd/C in 50 ml of MeOH is suspended in a 100 ml two-necked flask and saturated with hydrogen. Then, the solution of 595 mg [4] (1 mmol) in 5 ml of methanol and 1.6 ml of 6N hydrochloric acid is added with a one-way syringe by a septum. After completion of the absorption of hydrogen, it is separated from the catalyst and the solvent is drawn off in a vacuum.

White crystals remain. The recrystallization takes place from methanol.

Yield: 76% 1H-NMR data in DMSO/TMS 1.0 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 2.8 ppm (s,2H,CH2Ar); 4.2 ppm (m,4H,ArCH2NH); 7.0-7.8 ppm (m,10H,ArH) 2-Methvl-2-(4-isothiocyanatobenzyl)-N.N'-propylene-bis-i(5sulfo)salicylidenaminel Γ61 An 85% solution of thiophosgene in carbon tetrachloride (0.2 ml, 2.23 mmol) is added to the solution of 280 mg [5] (0.5 mmol) in 4 ml of hydrochloric acid (3 M). The reaction mixture is stirred for 3 hours at room temperature and then evaporated to dryness in a vacuum.

Yield: 78% 1H-NMR data in DMSO/TMS 1.0 ppm (s,3H,Me); 2.8 ppm (m,4H,CH2NH); 3.0 ppm (s,2H,CH2Ar); 4.2 ppm (m,4H,ArCH2NH); 7.0-7.8 ppm (m,10H,ArH) Example 2: 2-(4-Nitrobenzvl)-malonic acid diethyl ester Γ71 21.4 g of lithium diisopropylamide is added to 210 ml of anhydrous tetrahydrofuran under a nitrogen stream in a dry threenecked flask with a drying tube and dropping funnel with pressure compensation. Then, 58.0 g of malonic acid diethyl ester in 100 ml of anhydrous tetrahydrofuran is instilled at room temperature within 40 minutes. After 30 minutes, the reaction solution is cooled to -62°C and 39.1 g of 4-nitrobenzyl bromide in tetrahydrofuran is instilled slowly with vigorous stirring, it is stirred for another hour at -62°C and then the formed precipitate is filtered off cold. The filtrate is concentrated by evaporation on a rotary evaporator, the residue is brought into solution in 300 ml of ethanol at 65°C and separated from the insoluble residue. After cooling, 34.7 g of pale yellowish crystals are obtained.

Yield: 65% 1H-NMR data in CDC13/TMS 1.2 ppm (t,6H,CH2CH3); 3.3 ppm (d,2H,CH2Ar); 3.6 ppm (t,lH,CH); 4.1 ppm (q,4H,CH2CH3); 7.4 ppm (d,2H,ArH); 8.1 ppm (d,2H,ArH) 2-(4-Nitrobenzyl)-malonic acid diamide Γ81 This compound is produced corresponding to compound [2].

Yield: 98% ^-H-NMR data in DMSO/TMS 3.1 ppm (d,2H,CH2Ar); 3.4 ppm (t,lH,CH); 7.1 ppm (s,4H,NH2); 7.4 ppm (d,2H,ArH); 8.2 ppm (d,2H,ArH) 2-(4-Nitrobenzyl)-1,3-propanediamine f91 This compound is produced corresponding to compound [3].

Yield: 76% 1H-NMR data in D2O 1.3 ppm (t,lH,CH); 3.1 ppm (d,4H,CH2NH2); 3.2 ppm (d,2H,CH2Ar); 7.5 ppm (d,2H,ArH); 8.2 ppm (d,2H,ArH) 2-(4z-Nitrobenzyl)-N,Nz-propylene-bis-f(5sulfo)salicvlideniminel [101 A solution of 9.5 g of 5-sulfosalicylaldehyde in 75 ml of ethanol abs. is instilled in a solution of 5.6 g of diamine [9] in 75 ml of ethanol abs. with stirring. The resulting solution is slowly discolored intensively yellow. It is stirred for another 3 hours at room temperature, in this way, a more yellow precipitate precipitates, which is filtered off after cooling to -20°C.

Yield: 68% 1H-NMR data in DMSO/TMS 1.5 ppm (m,lH,CH); 3.2 ppm (d,2H,CH2Ar); 3.5 ppm (m,4H,CH2N=C); 7.1-7.9 ppm (m,8H,ArH); 8.2 ppm (d,2H,ArH); 8.6 ppm (s,2H,CH=N) 2— f4 z—Nitrobenzvll-N,Nz-propylene-bis-r(5-sulfo)salicylidenaminel mi 500 mg of sodium borohydride is added to a solution of 2 g of diimine [10] in 50 ml of 50% ethanol with stirring and stirred for 4 hours at 0°C. Then, 20 ml of water is instilled and stirred for 1 hour at room temperature, then the solvent is drawn off on a rotary evaporator and brought to pH 11 with a saturated potassium carbonate solution. The residue is taken up in methanol and filtered by a short column (RP-18). After drying on sodium sulfate and removal of the solvent, a crystalline residue remains.

Yield: 80% 1H-NMR data in DMSO/TMS 1.6 ppm (m,lH,CH); 2.5 ppm (m,4H,CH2NH); 2.9 ppm (d,2H,CH2Ar); 4.0 ppm (m,4H,ArCH2NH); 7.1-7.8 ppm (m,8H,ArH); 8.2 ppm (d,2H,ArH) Cu-2-(4'-Nitrobenzyl)-Ν,N'-propylene-bis-f(5sulfo)salicylidenamine] Γ121 400 mg of copper acetate (2 mmol) in 20 ml of methanol is instilled in a suspension of 1.63 g [11] (2 mmol) in 50 ml of methanol and stirred at room temperature. After removal of the solvent and drying, a crystalline residue remains.

Yield: 66% Cu-2-(4'-Aminobenzyl)-N.N'-propylene-bis-f(5sulfo)salicylidenamine] Γ13] This compound is produced corresponding to compound [5].

Yield: 78% Cu-2-f 4'-Biotincarbamovl)benzyl]-Ν.N'-propylene-bis-Γ(5sulfo)salicylidenamine1 Γ14] 680 mg of NHS-biotin (2 mmol) is added to a solution of 615 mg [13] (1 mmol) in 10 ml of DMSO with stirring and heated for 3 hours to 50°C. After cooling to room temperature, it is stirred for another 15 hours. Then, the solvent is drawn off in a vacuum and the residue is purified by MPLC (silica gel, dichloromethane/ethanol/concentrated ammonia 10:20:1) Yield: 38% - f 4'-(Biotincarbamoyl) benzyl1-N,Nz-propylene-bis-f(5sulfo)salicylidenamine) Γ151 500 mg of potassium cyanide is added to a solution of 500 mg [14] in 70 ml of water at 40°C and stirred for another 2 hours. Then, it is concentrated by evaporation to about 10 ml and the residue is purified by MPLC (silica gel, acetonitrile/concentrated ammonia 40:3).

Yield: 46% 1H-NMR data in DMSO/TMS 1.3-1.7 ppm (m,7H,CH2+CH); 2.3 ppm (t,2H,CH2CO); 2.8 ppm (m,2H,CH2S); 3.0 ppm (s,2H,CH2Ar); 3.1 ppm (m,lH,CHS); 4.1 ppm (m,1H,CHNHCO); 4.3 ppm (m,5H,ArCH2NH+CHNHCO); 6.9 ppm-7.8 ppm (m,10H,ArH).

Example 2a Tc-99m Complex of ί151 .6 * 10-3 mol of potassium tartrate in 1 ml of H2O, 4.8 * 10-8 mol of tin chloride in 1 ml of H2Q and 5 mCi of Tc-99m pertechnetate are added to a solution of 1.86 * 10“^ mol [15] in 1 ml of EtOH/H2O 1:9 and incubated for 10 minutes. The corresponding technetium complex is obtained in a purity greater than 90%.

Example 3 - (4'-Nitrobenzyl)-N,N'-propylene-bis-f (5carboxy)salicylidenaminel Γ161 This compound is produced corresponding to compound [4]. Yield: 66% 1H-NMR data in DMSO/TMS 0.9 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 2.8 ppm (s,2H,CH2Ar); 4.2 ppm (m,4H,ArCH2NH); 6.9-8.0 ppm (m,8H,ArH); 8.2 ppm (d,2H,ArH) Cu-2-(4'-Nitrobenzyl)-N,N'-propylene-bis-f(5carboxy)salicylidenaminel Γ171 This compound is produced corresponding to compound [12]. Yield: 74% Cu-2-(4'-Aminobenzyl)-N,Ν'-propylene-bis-Γ5(carboxy)salicylidenaminel Γ18Ί This compound is produced corresponding to compound [13].

Yield: 69% CU-2-T4'-(Biotincarbamoyl)benzyll-N,N'-propylene-bis-r (5carboxv)salicylidenaminel Γ191 This compound is produced corresponding to compound [14].

Yield: 43% 2-r4 z-(Biotincarbamoyl)benzyl]-N,NZ-propylene-bis-f(5carboxy)salicylidenaminel Γ201 This compound is produced corresponding to compound [15].

Yield: 49% 1H-NMR data in DMSO/TMS 1.3-1.7 ppm (m,7H,CH2+CH); 2.3 ppm (t,2H,CH2CO); 2.8 ppm (m,2H,CH2S); 3.0 ppm (s,2H,CH2Ar); 3.1 ppm (m,lH,CHS); 4.1 ppm (m,1H,CHNHCO); 4.2 ppm (m,5H,ArCH2NH+CHNHCO); 6.9 ppm-8.1 ppm (m,10H,ArH).

Example 4 2-Bromomethyl-5-nitrobenzoic acid Γ211 ml of bromine is slowly instilled in 72 g of 2-methyl-5nitrobenzoic acid and 100 mg of dibenzoyl peroxide in 250 ml of carbon tetrachloride under irradiation with a 500 W incandescent lamp. The reaction mixture is refluxed for 24 hours, the solvent is drawn off after cooling, taken up in dichloromethane, washed several times with water, dried and concentrated by evaporation. The residue is chromatographically purified (silica gel, dichloromethane).

Yield: 56% 1H-NMR data in CDC13/TMS 4.4 ppm (s,2H,CH2); 8.3-8.5 ppm (m,3H,ArH). (2-Carboxy-4-nitrobenzyl)malondinitrile f 221 The solution of 16.4 g of malondinitrile in 100 ml of anhydrous DMF is instilled in a suspension of 11.9 g of sodium hydride in 100 ml of anhydrous DMF at 0°C within 3 hours. Then, 65 g of [21] in 100 ml of anhydrous DMF is instilled within 6 hours and stirred after heating to room temperature for another 12 hours. The reaction mixture is hydrolyzed with ice water, acidified and extracted with chloroform. After drying and concentration by evaporation, a crystalline residue remains, which is recrystallized from methanol.

Yield: 48% 1H-NMR data in CDC13/TMS 3.5 ppm (d,2H,CH2Ar); 3.8 ppm (t,lH,CH); 8.3-8.5 ppm (m,3H,ArH). 2-(2-Carboxy-4-nitrobenzvl)-1.3-propanediamine r 231 g of sodium borohydride is slowly added to a solution of g of [22] (0.05 mol) and 24 g of cobalt chloride hexahydrate (0.1 mol) in 300 ml of 99% methanol at -15°C and then stirred for another 3 hours. After adding 100 ml of 3N hydrochloric acid, the solvent is distilled off for the most part, the remaining aqueous phase is alkalized with saturated potassium carbonate solution and extracted several times with dichloromethane. After dryinq and concentration by evaporation, a residue remains which is recrystallized from ethanol.

Yield: 59% 1H-NMR data in DMSO 1.3 ppm (t,lH,CH2); 3.1 ppm (d,2H,CH2NH2); 3.4 ppm (d,2H,CH2Ar); 8.3-8.5 ppm (m,3H,ArH) - Γ 2 z-Carboxy-(4'-nitrobenzyl)1-N,Nz-propylene-bissalicylidenamine Γ241 This compound is produced corresponding to compound [4].

Yield: 62% ^-H-NMR data in CDC13/TMS 1.6 ppm (m,lH,CH); 2.5 ppm (m,4H,CH2NH); 3.0 ppm (d,2H,CH2Ar); 3.9 ppm (m,4H,ArCH2NH); 6.9 ppm (m,4H,ArH); 7.1-7.3 ppm (m,4H,ArH); 8.3-8.4 ppm (m,3H,ArH) 2-Γ 2 z-Carboxy-(4 z-aminobenzyl)1-N,Nz-propylene-bissalicylidenamine Γ251 This compound is produced corresponding to compound [5].

Yield: 85% 1H-NMR data in DMSO/TMS 1.5 ppm (m,lH,CH); 2.5 ppm (m,4H,CH2NH2); 2.9 ppm (t,2H,CH2Ar); 4.1 ppm (m,4H,ArCH2NH); 6.8-7.4 ppm (m,llH,ArH) 2-Γ 2 z-Carboxy-(4 z-isothiocvanatobenzyl) 1-N,Nz-propylene-bissal icy lidenamine Γ261 This compound is produced corresponding to compound [6].

Yield: 77% ^H-NMR data in DMSO/TMS 1.5 ppm (m,lH,CH); 2.9 ppm (m,5H,CH2NH+CH2Ar); 3.0 ppm (s,2H,CH2Ar); 4.1 ppm (m,4H,ArCH2NH); 6.8-7.4 ppm (m,llH,ArH).

Example 5 6- f 2'-Carboxy-4'-nitrobenzyl)-3,3,9,9-tetramethyl-4,8diazaundecane-2,10-dione-dioxime Γ271 g of diamine [23] is dissolved in 90 ml of methanol and cooled to 0°C, then 6.5 g of 2-chloro-2-methyl-3-nitrosobutane is added with stirring. After heating to room temperature, it is stirred for 2 hours, refluxed for another 2 hours, the solvent is drawn off and the remaining solid is dissolved in water. It is neutralized with sodium bicarbonate, concentrated by evaporation, and the remaining residue is recrystallized from ethanol.

Yield: 38% 1H-NMR data in DMSO/TMS 1.3 ppm (s,12H,MeCNH); 1.8 ppm (s,6H,MeC=N); 2.4 ppm (m,lH,CH); 2.5 ppm (m,4H,CH2NH); 2.9 ppm (d,2H,CH2Ar); 8.2-8.4 ppm (m,3H,ArH); 10.8 ppm (s,2H,HON=C) 6- (2'-Carboxv-4'-aminobenzvl)-3.3,9,9-tetramethyl-4,8diazaundecane-2,10-dione-dioxime [281 500 mg of nitro compound [27] is dissolved in 50 ml of 50% methanol (pH 11) and hydrogenated at room temperature with adding 25 mg of Pd/Alox. After separating the catalyst and concentration by evaporation, a crystalline solid remains.

Yield: 49% 1H-NMR data in D2O 1.2 ppm (s,12H,MeCNH); 1.7 ppm (s,6H,MeC=N); 1.9 ppm (m,lH,CH); 2.5 ppm (m,4H,CH2NH); 2.5 ppm (d,2H,CH2Ar); 6.7 ppm (d,lH,ArH); 7.3 ppm (m,2H,ArH) Cu-6-(2'-Carboxy-4'-aminobenzyl)-3,3,9.9-tetramethyl-4,8diazaundecane-2,10-dione-dioxime f291 This compound is produced corresponding to compound [12].

Yield: 58% Cu-6-r 2'-Carboxy-4'-(biotincarbamovl) benzyl1-3,3,9,9-tetramethyl 4.8-diazaundecane-2,10-dione-dioxime Γ301 This compound is produced corresponding to compound [14].

Yield: 34% 6-(2'-Carboxv-4'-biotincarbamovl)benzyl1-3,3,9,9-tetramethyl-4,8 diazaundecane-2,10-dione-dioxime Γ 311 This compound is produced corresponding to compound [15].

Yield: 41% Tc-99m Complex of [31] 3.2 * 10~3 mol of potassium tartrate in 1 ml of H2O, 4.0 * 108 mol of tin chloride in 1 ml of H2O and 5 mCi of Tc-99m pertechnetate are added to a solution of 1.86 * 10-8 mol of [31] in 1 ml of EtOH/H2O 1:4 and incubated for 10 minutes. The corresponding technetium complex is obtained in a purity greater than 90%.

Example 6 2-Methyl-2-Γ4'-nitrobenzoyll-malonic acid dimethyl ester [321 ml of carbon tetrachloride is added to the mixture of 24.3 g of magnesium chips (1 mol) in 50 ml of anhydrous ethanol.

Then, a solution of 174 g of methylmalonic acid diethyl ester (1 mol) in 100 ml of ethanol and 400 ml of anhydrous ether is instilled so that the mixture boils vigorously. After dissolution of the magnesium, 185 g of 4-nitrobenzoyl chloride in 100 ml of anhydrous ether is instilled under ice cooling and stirred for another 12 hours. It is hydrolyzed under ice cooling, the ether phase is separated, extracted several times with ether, washed neutral, dried and concentrated by evaporation. After distillation in a vacuum, the pure product is obtained.

Yield: 78% 1H-NMR data in CDC13/TMS 1.2 ppm (m,9H,Me); 4.1 ppm (q,4H,CH2Me); 8.1-8.3 ppm (m,4H,ArH) 2-Methyl-2-Γ4z-nitrobenzoyll-malonic acid Γ331 g of [32] (127 mmol) is dissolved in 200 ml of methanol and a solution of 28.3 g of NaOH (708 mmol) in 50 ml of water is slowly instilled in it. Then, it is heated for 2 hours in a water bath to 50°C, the solvent is drawn off on a rotary evaporator and the residue is taken up in water. It is acidified with semiconcentrated hydrochloric acid and the free carboxylic acid is extracted with chloroform, washed and dried.

Yield: 80% 1H-NMR data in DMSO/TMS 1.2 ppm (s,3H,Me); 8.1-8.3 ppm (m,4H,ArH) 2-Methvl-2-r4'-nitrobenzoyl]-malonic acid chloride Γ341 The mixture of 3.40 g of [33] and 8.9 g of thionyl chloride and one drop of DMF is heated to boiling under exclusion of moisture with stirring. After completion of the gas generation, the excess thionyl chloride is drawn off at room temperature in a vacuum and the residue is distilled in a vacuum.

Yield: 91% N.N'-bisf(2-(Benzylthio)benzyl1-2-methyl-2-(4'nitrobenzoyl) malonic acid diamide [35] 7.4 g of [34] (24.3 mmol) in 100 ml of dichloromethane is carefully instilled in the mixture of 10.5 g of (2mercaptobenzyl)benzylamine (48.6 mmol) and 5.06 g of triethylamine (50 mmol) in 100 ml of dichloromethane with exclusion of moisture. It is stirred for another 3 hours at room temperature, then first water is added and extracted several times with chloroform. The organic phase is washed in succession with IN hydrochloric acid, saturated potassium carbonate solution and water and dried on sodium sulfate.

Yield: 64% 1H-NMR data in CDC13/TMS 1.2 ppm (s,3H,Me); 4.1 ppm (s, 8H, ArCI^S+ArC^NH) ; 7.17.6 ppm (m,18H,ArH); 8.3 ppm (m,4H,ArH).

N,Nz-bis Γ(2-Benzylthio)benzyl 1-2-methyl-2-Γhydroxy-(4nitrophenyl)-methyl)-malonic acid diamide Γ36) .7 g of sodium borohydride is added to a solution of 66 g (100 mmol) of [35] in 50 ml of ethanol and stirred for 2 hours at room temperature. It is substantially concentrated by evaporation and the residue is mixed with 50 ml of water and neutralized. It is extracted with ether, washed with saturated common salt solution, dried and concentrated by evaporation.

Yield: 77% 1H-NMR data in DMSO/TMS 1.4 ppm (s,3H,Me); 4.1 ppm (s,8H,ArCH2S+ArCH2NH); 4.3 ppm (m,lH,CHOH); 7.1-7.6 ppm (m,20H,ArH); 8.2 ppm (m,2H,ArH).

N,Nz-bisf(2-Benzylthio)benzyl1-2-methyl-2-Γhydroxy-(4nitrophenvl)methyl)1-1,3-propanediamine Γ371 This compound is produced corresponding to compound [3]. 1H-NMR data in CDCl3/TMS 1.5 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 4.1 ppm (s,4H,ArCH2NH); 4.3 ppm (m,lH,CHOH); 7.0-7.5 ppm (m,10H,ArH); 8.2 ppm (d,lH,ArH).

N.Nz-bis(2-Mercaptobenzyl)-2-methyl-2-Γhydroxy-(4nitrophenvl)methyl)1-1,3-propanediamine Γ381 3.5 g of sodium is carefully added to a solution, cooled to -50°C, of 4.3 g of [37] in 50 ml of THF and 100 ml of liquid ammonia and stirred for 4 hours. Excess sodium is carefully !E 920761 destroyed with ammonium chloride and then allowed to heat slowly to room temperature. The residue is taken up in water and extracted several times with dichloromethane, the organic phase is dried and concentrated by evaporation.

Yield: 62% 1H-NMR data in CDC13/TMS 1.5 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 4.1 ppm (s,4H,ArCH2NH); 4.3 ppm (m,lH,CHOH); 7.0-7.5 ppm (m,10H,ArH); 8.2 ppm (d,1H,ArH).

N, Ν'—bis (2-Mercaptobenzvl)-2-methyl-2-[hydroxy-(4aminophenvl)methyl)1-1,3-propanediamine Γ391 This compound is produced corresponding to compound [5].

Yield: 89% 1H-NMR data in CDC13/TMS 1.5 ppm (s,3H,Me); 4.1 ppm (s,4H,ArCH2NH); 4.3 ppm (m,lH,CHOH); 7.0-7.7 ppm (m,llH,ArH).

N. Ν'-bis f 2-Mercaptobenzvl)-2-methvl-2-Γhydroxy-(4isothiocyanatophenvDmethvl)1-1,3-propanediamine Γ401 This compound is produced corresponding to compound [6].

Yield: 49% 1H-NMR data in CDC13/TMS 1.5 ppm (s,3H,Me); 4.1 ppm (s,4H,ArCH2NH); 4.2 ppm (m,lH,CHOH); 7.0-7.6 ppm (m,llH,ArH).

Example 7 N, Nz-bisΓ f 2-Benzylthio-3-pvridvl)methyl1-2-methyl-2-(4'nitrobenzyl)malonic acid diamide Γ411 This compound is produced corresponding to compound [35] Yield: 60% 1H-NMR data in CDC13/TMS 1.2 ppm (s,3H,Me); 3.5 ppm (s,2H,CH2Ar); 4.1 ppm (s,8H,ArCH2S+ArCH2NH); 7.1-7.5 ppm (m,14H,ArH); 8.3-8.5 ppm (m,6H,ArH).

N,Nz-bis Γ(2-Benzylthio-3-pyridyl)methyl1-2-methyl-2-(4 znitrobenzyl)-1,3-propanediamine ί421 This compound is produced corresponding to compound [3]. Yield: 57% ^•H-NMR data in CDC13/TMS 1.0 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 2.9 ppm (s,2H,CH2Ar); 4.1 ppm (s,8H,ArCH2NH+ArCH2S); 7.1-7.5 ppm (m, 14H,ArH) ; 8.3-8.5 ppm (m,6H,ArH).

N,Nz-bis Γ(2-Mercapto-3-pyridyl) methyl1-2-methyl-2-(4nitrobenzyl)-1,3-propanediamine [431 This compound is produced corresponding to compound [38] Yield: 65% 1H-NMR data in CDC13/TMS 1.0 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 2.9 ppm (s,2H,CH2Ar); 4.1 ppm (s,4H,ArCH2NH); 7.0-7.5 ppm (m,4H,ArH); 8.2-8.5 ppm (m,6H,ArH).

N,Nz-bis Γ(2-Mercapto-3-pyridvl) methyl 1-2-methyl-2-(4aroinobenzyl)-1,3-propanediamine Γ44 1 This compound is produced corresponding to compound [5]. Yield: 89% 1H-NMR data in CDCl3/TMS 1.0 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 2.8 ppm (s,2H,CH2Ar); 4.1 ppm (s,4H,ArCH2NH); 7.0-7.7 ppm (m,6H,ArH); 8.3-8.5 ppm (m,4H,ArH).

N,N'-bis Γ(2-Mercaptopyridyl)methyl1-2-methyl-2-(4isothiocvanatobenzyl)-1,3-propanediamine Γ451 This compound is produced corresponding to compound [6]. Yield: 46% 1H-NMR data in CDCl3/TMS 1.0 ppm (s,3H,Me); 2.5 ppm (m,4H,CH2NH); 2.8 ppm (s,2H,CH2Ar); 4.1 ppm (s,4H,ArCH2NH); 7.0-7.7 ppm (m,6H,ArH); 8.3-8.5 ppm (m,4H,ArH).

Example 8 Coupling and labeling of a chelating agent containing isothiocyanate to proteins The coupling of Tc-chelating agents containing isothiocyanate of compound [6] to proteins is to be described by the example of F(ab)2 fragments of monoclonal antibody 17-1A. Instead of antibody fragments, any other protein or a substance containing amino groups can be used.

Monoclonal antibody 17-1A is obtained according to methods known in the literature after administration of 10^ of the corresponding hybridoma cells in the abdominal space of a BALB/cmouse and aspiration of ascitic liquid after 7-10 days. The purification takes place also according to methods known in the literature by precipitation of ammonium sulfate and affinity chromatography by protein A-Sepharose. The purified antibody (10 mg/ml) is treated at pH 3.5 for 2 hours with 25 micrograms/ml of pepsin and then isolated by FPLC. Before the coupling with the chelating agent, the fragments are dialyzed at 4°C for 12-24 hours from 0.1 M of KH2PO4/0.1 M NaHCO3, pH 8.5 The protein concentration is adjusted to 10 mg/ml. A 5-fold molar excess of the chelating agent containing NCS [example 1] is dissolved in as little as possible of the same buffer and added to the protein solution. For conjugate formation, the mixture is incubated for 3 hours at 37°C. Then, the conjugate is dialyzed for 24-48 hours with repeated buffer change from PBS (phosphate-buffered saline) and the protein concentration is then, if necessary, adjusted again to 10 mg/ml. Until labeling with Tc-99m, the conjugate can be stored after sterilization by filtration at 4°C in acidpurified glass vessels.

The labeling of 1 mg of the antibody fragment with Tc-99m coupled with chelating agent [6] takes place by adding 10 mCi of pertechnetate solution (= 1-2 ml) and 100 micrograms of tin(II) chloride in an argon-flushed Na-pyrophosphate solution (1 mg/ml) or by ligand exchange, e.g., by adding the solution of a commercially available glucoheptonate kit mixed with pertechnetate.

Claims

Claims Compounds of general formula I in which R 1 stands for hydrogen or a C 1 _ g alkyl radical optionally substituted with one or two hydroxyl groups, R 2 stands for a C 1-6 alkylene radical optionally substituted with a hydroxyl group, R 3 stands for a hydrogen atom, a C 1 _ 6 alkyl radical, a carboxymethyl radical or a (C-^-θ alkoxycarbonyl) methyl radical, R 4 stands for a hydrogen atom, a alkyl radical optionally substituted with a hydroxyl group or' for the meaning indicated under R x , R 5 stands for a hydrogen atom or a C^^-θ alkyl radical optionally substituted with a hydroxyl group, B stands for a pyrrolyl radical, a substituted phenyl radical of formula II or a substituted pyridine radical of formula III in which Z means a hydroxyl group, amino group or a mercapto group and Y means a hydrogen atom, a carboxy radical or a sulfonyl radical or B stands for a nitrosomethyl radical of formula IV NOH R* x 4 in which R means a C^_g alkyl radical or, together with R , is a trimethylene or tetramethylene group to form a 5- or 6- membered ring and A stands for an amino radical, a mercapto radical, a carboxy radical, a C 2-6 alk i n y! or c 2-6 al ^ en Y 1 radical, an oxiranyl radical, a fluorinated phenoxycarbonyl radical, a succinimidoxycarbonyl radical optionally substituted with a sodium sulfate radical, an aminophenyl radical or isothiocyanatophenyl radical, and the phenyl group optionally can be substituted in addition by a carboxy radical, a chlorosulfonyl radical or a sulfonic acid radical or contains a compound T selectively accumulating in lesions or specific tissues — optionally bound by a bifunctional linker radical L with the help of said functional group and T stands for monoclonal antibodies or their fragments, hormones, enzymes, ligands for cell membrane receptors, neurotransmitters, lipids, steroids, saccharides, amino acids and oligopeptides, biotin, as well as radiosensitizers, such as, e.g., misonidazole, NOH Ii and in the case of B = -C, pyrrolyl radical or L R x Y = H, R 2 is to stand for a hydroxylated C 1-6 alkylene chain or A is to stand for a substituted aminophenyl or isothiocyanatophenyl radical, as well as their complexes with radioactive metal ions — suitable for diagnosis and treatment of tumors — as well as their salts with inorganic and organic acids or bases. I
2. Compounds according to claim 1, wherein radical L optionally contained in A has been produced from bifunctional linkers such as, e.g.,
3. Compounds according to claim 1, wherein T stands for monoclonal antibodies, their fragments, biotin or misonidazole.
4. Metal chelates according to claim 1 with coordinatively bound, radioactive ions of Tc, Re, Cu, Co, Ga, Y and In, preferably Tc and Re.
5. Use of the chelates according to claim 4 for in vivo diagnosis and treatment of tumors.
6. Pharmaceutical agents containing at least one chelate according to any of claims 1 to 5 optionally with the additives usual in galenicals.
7. Process for the production of compounds of general formula I in which R 1 stands for hydrogen or a alkyl radical optionally substituted with one or two hydroxyl groups, R 2 stands for a C-^g alkylene radical optionally substituted with a hydroxyl group, R 3 stands for a hydrogen atom, a Cj_g alkyl radical, a carboxymethyl radical or a (C^-g alkoxycarbonyl)methyl radical, R 4 stands for a hydrogen atom, a C-^-θ alkyl radical optionally substituted with a hydroxyl group or for the meaning indicated under R x , r5 stands for a hydrogen atom or a C^-g alkyl radical optionally substituted with a hydroxyl group, B stands for a pyrrolyl radical, a substituted phenyl radical of formula II or a substituted pyridine radical of formula III in which Z means a hydroxyl group, amino group or a mercapto group and Y means a hydrogen atom, a carboxy radical or a sulfonyl radical or B stands for a nitrosomethyl radical of formula IV NOH R* (IV) in which R x means a C 3 _g alkyl radical, which optionally is cyclized together with R 4 by a trimethylene or tetramethylene group to a 5- or 6-ring and A stands for an amino radical, a mercapto radical, a carboxy radical, a C 2 _g alkinyl or C 2 _ 6 alkenyl radical, an oxiranyl radical, a fluorinated phenoxycarbonyl radical, a succinimidoxycarbonyl radical optionally substituted with a sodium sulfate radical, an aminophenyl radical or isothiocyanatophenyl radical, and the phenyl group optionally can be substituted in addition by a carboxy radical, a chlorosulfonyl radical or a sulfonic acid radical or contains a compound T selectively accumulating in lesions or specific tissues — optionally bound by a bifunctional linker radical L with the help of said functional group and T stands for monoclonal antibodies or their fragments, hormones, enzymes, ligands for cell membrane receptors, neurotransmitters, lipids, steroids, saccharides, amino acids and oligopeptides, biotin, as well as radiosensitizers, such as, e.g., misonidazole, NOH Ii and in the case of B = -C, pyrrolyl radical or Y = H, R 2 is to stand for a hydroxylated C-|__ 5 alkylene chain or A is to stand for a substituted aminophenyl or isothiocyanatophenyl radical, as well as their complexes with radioactive metal ions — suitable for diagnosis and treatment of tumors — as well as their salts with inorganic and organic acids, wherein a) a 1,3-propanediamine of general formula V in which R 1 and R 2 have the above-mentioned meanings, and A' means A or a radical able to be converted to A is reacted with a compound of general formula VI B - R 4 C = Ο (VI) in which R 4 and B have the above-mentioned meanings, in a polar solvent, preferably ethanol, or by using a water separator in a nonpolar solvent, preferably benzene, at temperatures of 25-180°C within 6 hours to 3 days, the imino function is reduced in a way known in the art, preferably with sodium borohydride in a polar solvent, preferably a methanol/water mixture, at temperatures of 25-100°C within 0.5 to 24 hours, preferably 2 hours, or b) by a propanediamine of general formula V (V) in which R 1 , R 2 and A' have the above-mentioned meanings, being reacted with a compound of general formula VII B - CO-X (VII), in which B has the above-mentioned meaning, functional groups contained in B optionally are present in protected form and X stands for a halogen atom, preferably for a chlorine atom, or c) by a propanediamine of general formula V (V) in which R 1 , R 2 being reacted with a R 4 and A' have the above-mentioned meanings, compound of general formula VIII B—-C—R 5 X (VIII) in which R 4 , R^, b and X have the above-mentioned meanings and functional groups contained in B optionally are present in protected form, or d) substituted malonic acid halides, preferably malonic acid chlorides of general formula IX (IX) XOC cox I in which R 1 , R 2 , A' and X have the above-mentioned meanings and functional groups contained in B optionally are present in protected form, being reacted with an amine of general formula X R 4 I B—C—R 5 1 i' NHR J (X) in which R 4 , R 5 and B have the above-mentioned meanings, R 3 stands for a hydrogen atom or for a C 1-6 alkyl radical and functional groups contained in B optionally are present in protected fora, in an aprotic solvent, preferably dichloromethane, at temperatures of 0-180°C, preferably at room temperature, within 2 to 24 hours, preferably 4 hours, optionally by adding suitable bases, e.g., triethylamine, the amide function obtained according to process d) being reduced in a way known in the art, preferably with borane in THF or with lithium aluminum hydride in an aprotic solvent, preferably diethyl ether, at temperatures of 25-150°C within 0.5 to 24 hours, preferably
8. Hours to the corresponding amino function, the amino groups, in the case that R 3 ' stands for a hydrogen atom, optionally being alkylated in a way known in the art with an alkylating agent introducing R 3 and present protecting groups being cleaved and in the thus obtained compounds, group A 7 optionally converted to A — optionally after protecting the free amino groups and functional groups Z with protective ions, e.g., as a Cu complex — being generated and then optionally the thus obtained compounds being coupled by this functional group to selectively accumulating compounds T and substituents B optionally being complexed with the radioactive isotope desired in each case, and protective ions optionally present in the product in advance being removed according to methods known in the literature and the sequence of the steps of complexing with radioactive isotopes and coupling to T being able to be interchanged. IE 920761 -528. A compound substantially as hereinbefore described with reference to the Example.
9. A use substantially as hereinbefore described with reference to the Examples.
10. A pharmaceutical agent substantially as hereinbefore described with reference to the Examples.
11. A process substantially as hereinbefore described with reference to the Examples.