EP0851770A2

EP0851770A2 - Bifunctional sulphide-containing sulphonamide s 2?ny-type chelate-forming agents for radioisotopes

Info

Publication number: EP0851770A2
Application number: EP96945141A
Authority: EP
Inventors: Ludger Dinkelborg; Christoph Stephan Hilger; Wolfgang Kramp; Johannes Platzek; Bernd Radüchel; Sebastian Erber
Original assignee: Institut fuer Diagnostikforschung GmbH
Current assignee: Bayer Pharma AG
Priority date: 1995-09-21
Filing date: 1996-09-19
Publication date: 1998-07-08
Also published as: WO1997012636A2; AU1436197A; DE19536785A1; WO1997012636A3; CA2232315A1

Abstract

The invention concerns novel compounds of general formula (I), M-L, in which M stands for a radioisotope of Tc or Re and L stands for a ligand of general formula (II) R?1S-CR2R3-(CR4R5)¿n=1,2-S-CHR?6-CHR7-SO¿2-NH-(CR8R9)m=1,2-CO-B in which R?1, R2, R3, R4, R5, R6, R7, R8, and R9¿ can have different meanings and B stands for a further group which is suitable for the dative bond of metal ions and for the coupling to selectively concentrating compounds. The novel compounds are used for complexing technetium and rhenium and are applied in medical diagnostics and therapy.

Description

Bifunctional sulfide-containing sulfonamide chelating agents of type S ₂ NY for radioactive isotopes

The invention relates to new chelating agents containing sulfonamide groups, pharmaceutical compositions containing these compounds, their use in radiodiagnostics and radiotherapy, processes for the preparation of these compounds and compositions, and conjugates of these compounds with substances which accumulate selectively in diseased tissue, in particular peptides.

The use of radio pharmaceuticals for diagnostic and therapeutic purposes has long been known in the field of biological and medical research

In particular, radiopharmaceuticals are used to represent certain structures such as the skeleton, organs or tissues. The diagnostic application presupposes the use of such radioactive agents, which are specific to those after application

Enrich structures in the patient to be examined. These locally enriching radioactive agents can then be tracked down, plotted or scanned using suitable detectors, such as, for example, imaging cameras or other suitable recording methods. The distribution and relative intensity of the detected radioactive agent characterizes the location of a structure in which the radioactive agent is located and can be the presence of abnormalities in structure and function, pathological

Represent changes etc. Similarly, radiopharmaceuticals can be used as therapeutic agents to irradiate certain pathological tissues or areas. Such treatment requires the production of radioactive therapeutic agents that accumulate in certain structures, tissues or organs. By enriching these agents, the therapeutic radiation is carried directly to the pathological tissue.

The use of both diagnostic and therapeutic radiopharmaceuticals requires radio-labeled compounds. In the case of metallic radionuclides, the metal can be in free form as an ion or in the form of a metal complex with one or more ligands. Examples of metallic radionuclides that can form complexes are technetium-99m and the various rhenium isotopes. The first is used in diagnostics and the second in therapy. The radiopharmaceuticals contain suitable carriers and additives which allow injection, inhalation or ingestion by the patient, as well as physiological buffers, salts etc.

The most frequently used radionuclide for nuclear medicine questions is technetium-99m, which due to its favorable physical properties (no corpuscular radiation, 6 h physical half-life, 140 KeV gamma radiation) and the resulting low radiation exposure is particularly good as a radioisotope for in vivo Diagnostics are suitable.

Technetium-99m can be easily obtained from nuclide generators as pertechnetate and can be used directly in this form for the production of kits for routine clinical needs.

The production of radiopharmaceuticals first requires the synthesis of a suitable ligand. Then the complex with the radionuclide is shown separately (marking). The ligand produced, always in the form of a lyophilized kit, is mixed with a solution containing the radionuclide Complex formation conditions implemented. If, for example, the production of a technetium-99m radiopharmaceutical is desired, the ligand produced is mixed with a pertechnetate solution with the addition of a suitable reducing agent and the corresponding technetium complex is produced under suitable reaction conditions. These complexes are then suitably administered to the patient by injection, inhalation or ingestion.

The solutions containing the radionuclide can be obtained from an available Mo-99 / Tc-99m nuclide generator, as in the case of technetium-99m, or from a manufacturer, as in the case of rhenium-186. The complex formation reaction is carried out at suitable temperatures (e.g. 20 ° -100 ° C) within a few minutes to several hours. In order to ensure complete complex formation, a large excess (more than 100-fold excess to the metal radionuclide) of the ligand produced and a sufficient amount of reducing agent are required for a complete reduction of the radionuclide used.

Radiopharmaceuticals are made by combining the radionuclide complex, in an amount sufficient for diagnostic or therapeutic use, with pharmacologically acceptable radiological carriers. This radiological carrier should have favorable properties for the application of the radiopharmaceutical in the form of an injection, inhalation or ingestion. Examples of such carriers are HSA, aqueous buffer solutions, for example tris (hydroxymethyl) aminoethane (or their salts), phosphate, citrate, bicarbonate etc., sterile water, physiological saline, isotonic chloride or dicarbonate Ion solutions or normal plasma ions such as Ca ²⁺ , Na ⁺ , K ⁺ and Mg2 ⁺ .

Since technetium can exist in a number of oxidation states (+7 to -1), it is often necessary for radiopharmaceutical agents to contain additional agents known as stabilizers. These keep the radionuclide in a stable form until it has reacted with the ligand. These stabilizers can include agents known as transfer or auxiliary ligands that are particularly useful in stabilizing and complexing the metal in a well-defined oxidation state until the target ligand complexes the metal via ligand exchange. Examples of this type of auxiliary ligand are

(including their salts) gluconheptonic acid, tartaric acid, citric acid, or other common ligands, as detailed below.

Radionuclide is the standard

Radiopharmaceuticals are shown by first synthesizing the ligand and then reacting it with the metal radionuclide in a suitable manner in order to form a corresponding complex in which the ligand must necessarily be present unchanged after complexation, with the exception of the splitting off of any protective groups or hydrogen ions that may be present. Removal of these groups facilitates coordination of the ligand to the metal ion and thus leads to rapid complexation.

To form technetium-99m complexes, pertechnetate is first obtained from a nuclide generator and converted to a lower oxidation level by using suitable reducing agents (eg SnCl2, S2O4 ^{2 "} etc.), which is then converted by a suitable one

Chelator is stabilized. Because technetium in a row of oxidation levels (+7 to -1), which can change the pharmacological properties by changing the charge of a complex, it is necessary to provide chelators or complex ligands for technetium-99m, which technetium safely, firmly and stably in a defined Can bind oxidation level in order to prevent undesired biodistribution taking place due to redox processes or technetium releases from the corresponding radio diagnostics, which complicates the reliable diagnosis of corresponding diseases.

The efficiency of radionuclides in in vivo diagnostics as well as therapy depends on the specificity and the selectivity of the labeled chelates to the target cell. These properties can be improved by coupling the chelates to biomolecules according to the "drug targeting" principle. Antibodies, their fragments, hormones, growth factors and substrates of receptors and enzymes are suitable biomolecules. The British patent application GB 2,109,407 describes the use of radioactively labeled monoclonal antibodies against tumor-associated antigens for tumor diagnosis in vivo. Likewise, direct

Protein labeling via donor groups (amino, amide, thiol, etc.) of the protein (Rhodes, BA et al., J. Nukl. Med. 1986, 27, 685-693) or by introducing complexing agents (US Patent 4,479,930 and Fritzberg, AR et al. Nucl. Med. 1986, 27, 957) with technetium-99m. However, these experimental methods are not available for clinical use because on the one hand the selectivity is too low and on the other hand the background activity is too high in the organism to enable in vivo imaging. Suitable complexing agents for technetium and rhenium isotopes are, for example, cyclic amines as described by Volkert et al. (Appl. Radiol. Isot. 1982, 33; 891) and Troutner et al. (J. Nucl. Med. 1980, 21; 443), but they have the disadvantage that they are often only able to bind technetium-99m in good yields from a pH> 9. N2θ2 systems (Pillai, MRA, Troutner, DE et al.; Inorg. Chem. 1990, 29; 1850) are in clinical use. A major disadvantage of non-cyclic N ₄ systems such as HMPAO is their low complex stability. Tc-99m-HMPAO must because of its instability (Ballinger, JR et al., Appl. Radiat. Isot. 1991, 42; 315), Billinghurst, MW et al. , Appl. Radiat. Isot. 1991, 42; 607) can be applied within 30 minutes after its labeling, so that the proportion of decay products that have a different pharmacokinetics and excretion can be kept low. Such radiochemical contaminants make it difficult to identify diseases to be diagnosed. A coupling of these chelates or chelating agents to other substances that selectively accumulate in foci of disease cannot be solved with simple means, so that they are generally distributed nonspecifically in the organism.

N ₂ S2 chelators (Bormans, G. et al .; Nucl. Med. Biol. 1990, 17; 499) such as, for example, ethylenedicysteine (EC; Verbruggen, AM et al .; J. Nucl. Med. 1992, 33; 551 ) meet the requirement for sufficient stability of the corresponding technetium-99m complex, but only form> 9 radio diagnostics with a purity of greater than 69% from a pH value of the complexing medium. N3S systems (Fritzburg, A.; EP-0173424 and EP-0250013) form stable technetium-99m complexes, but have to be heated to temperatures of approx. 100 ° C to form a uniform radiopharmaceutical. In recent years, the need for radio-diagnostic agents specifically enriching in diseased tissues has increased. This can be achieved if complexing agents can easily be coupled to selectively enriching substances and do not lose their favorable complexing properties. However, since it often happens that after coupling a complexing agent using one of its functional groups to such a molecule, a weakening of the complex stability is observed, the previous approaches for coupling chelating agents to substances that selectively enrich themselves are not very satisfactory, since a diagnostically not tolerable proportion of the isotope from the conjugate is released in vivo (Brechbiel, MW et al.; Inorg. Chem. 1986, 25, 2772). It is therefore necessary to present bifunctional complexing agents which carry both functional groups for binding the desired metal ion and one (other, several) functional group for binding the selectively enriching molecule, or to design complexing agents in such a way that they are only selectively coupled to one enriching substance the desired complexing agent structure is formed and thus a weakening of the complex stability is excluded. Such ligands enable a specific, chemically defined binding of technetium or rhenium isotopes to various biological materials, even if so-called prelabeling is carried out. Some chelating agents coupled to monoclonal antibodies (eg EP-0247866 and EP-0188256) or fatty acids (EP-0200492) have been described. However, the previously mentioned N2S2 systems are used as chelating agents, which are not very suitable due to their low stability. Because both the selectively enriching substances in their properties, as well as the Mechanisms according to which they are enriched are very different, it is still necessary to vary the coupling-capable chelating agent and to be able to adapt it to the physiological requirements of the coupling partner with regard to its lipophilicity, membrane permeability, etc.

The invention is therefore based on the object of providing stable complex compounds which are coupled or capable of coupling to different selectively enriching compounds without their specificity and selectivity being significantly affected. In addition, there is the task of providing couplable chelators or complexes which have a greater chemical range of variation of the substituents in order to be able to adapt them to the requirements referred to above. At the same time, the requirements for the use of these compounds in humans with regard to the absorbed radiation dose, stability and solubility of the compounds must be fulfilled.

According to the invention, this object is achieved in that new chelating agents containing bifunctional sulfonamide groups and their coupling products with specifically enriching compounds are made available.

The invention relates to compounds of the general formula (I)

M - L (I)

wherein M is a radioisotope of Tc or Re and L is a ligand of the general formula (II) Rl-S-CR ² !. ³ - (CR ⁴ R ⁵ ) _{n = 1 # 2} -S-CHR ⁶ -CHR ⁷ -S0 ₂ -NH- (CR ⁸ R ⁹ ) _{m = 1/2} -CO-B

(II) means where

R ^{1 represents} a hydrogen atom, a branched or unbranched C _] __g alkyl radical or a sulfur protecting group,

R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ - R ⁸ and R ^{9 are the} same or different and each represents a hydrogen atom and / or a branched or unbranched C _] __g alkyl radical,

B for a hydroxyl group, a mercapto group or a radical -NHR ^a , wherein

R ^{a is} a hydrogen atom, a branched or straight-chain, cyclic or polycyclic ^c 1-30 alkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, aryl, alkylaryl or

Arylalkyl radical, which is optionally substituted with hydroxy, oxy, oxo, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde or alkoxy groups with up to 20 carbon atoms and / or optionally with one or more heteroatoms from the O series , N, S, P, As, Se is interrupted and / or substituted.

Preferred compounds of the general formula (I) are characterized in that n and m each represent 1 and that R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and R ^{9 are} hydrogen atoms. Particularly preferred compounds of the general formula

(I) are characterized in that n and m each represent 1 and that R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and R ^{9 are} hydrogen atoms and B represents a hydroxyl group or a radical -NHR ^a , wherein

R ^{a is} a hydrogen atom, a branched or straight-chain cyclic or polycyclic C _] __3 _Q - alkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, aryl, alkylaryl or arylalkyl radical, optionally with hydroxy, oxy, oxo -, Carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde or alkoxy groups with up to 20 carbon atoms and / or optionally interrupted by one or more heteroatoms from the series 0, N, S, P, As, Se and / or is substituted.

The invention further relates to the new bifunctional sulfur-atom-interrupted sulfonamide ligands of the general formula (II)

R ¹ S-CR ² R ³ - (CR ⁴ R ⁵ ) _{n = 1 # 2} -S-CHR ⁶ -CHR ⁷ -S0 ₂ -NH- (CR ⁸ R ⁹ ) _{m = 1 f 2} -CO-B

(II)

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ 'R ⁷ , R ⁸ , R ⁹ , n, m and B each have the meaning given above.

Preferred compounds of the general formula (II) are distinguished in that n and m each represent 1 and in that R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and R ^{9 are} hydrogen atoms.

Particularly preferred compounds of the general formula (I) are characterized in that 1 for each of n and m is and that R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and R ^{9 are} hydrogen atoms and B is a hydroxyl group or a radical -NHR ^a , wherein

R ^{a is} a hydrogen atom, a branched or straight-chain cyclic or polycyclic C _] __3 _Q - alkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, aryl, alkylaryl, arylalkyl radical, optionally with hydroxy, oxy, oxo -, Carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde or alkoxy groups with up to 20 carbon atoms and / or optionally interrupted by one or more heteroatoms from the series O, N, S, P, As, Se and / or is substituted.

The invention further relates to conjugates containing a compound of the general formula (I and / or II) and nucleotides of the DNA and RNA type, and also selectively accumulating in diseased tissue

Substances with a convex bond between them and, in the case of substances containing carboxyl or amino groups, such as naturally occurring or modified oligonucleotides in which the degradation is prevented or made more difficult by naturally occurring nucleases, peptides, proteins, antibodies or fragments thereof amidically or is ester-like in the case of hydroxyl-containing substances such as fatty alcohols or imidic in the case of substances containing aldehyde groups.

Particularly preferred conjugates are characterized in that the substances accumulating in the diseased tissue are peptides such as endotheline, partial sequences of endothelin, endothelin analogues, endothelin derivatives,

Endothelin antagonists or angiotensins, partial sequences of angiotensins, angiotensin analogs, angiotensin derivatives and angiotensin antagonists as well as chemotactic peptides.

In further preferred conjugates according to the invention, the peptides have the following sequences

Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr I ₁

Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Ser-Cys-Ser-Ser-Trp-Leu-Asp-Lys-Glu-Cys-Val-Tyr-

Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Thr-Cys-Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Cys-Val-Tyr-1

Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Ser-Ala-Ser-Ser-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-

Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Ser-Cys-Lys-Asp-Met-Thr-Asp-Lys-Glu-Cys-Leu-Asn-

Phe-Cys-His-Gln-Asp-Val-Ile-Trp,

I i

Ala-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-

Phe-Ala-His-Leu-Asp-Ile-Ile-Trp, Ala-Ser-Ala-Ser-Ser-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr- Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,

Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr- Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Thr-Cys-Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Ala-Val-Tyr- Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,

Cys-Val-Tyr-Phe-Cys-His-Gin-Asp-Val-Ile-Trp,

N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Gin Asp-Val-Ile-Trp,

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,

Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe,

Arg-Val-Tyr-Ile-His-Pro-Phe,

Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,

Sar-Arg-Val-Tyr-Val-His-Pro-Ala,

For-Met-Leu-Phe,

For-Met-Leu-Phe-Lys,

the partial sequences

His-Leu-Asp-Ile-Ile-Trp,

D-Trp-Leu-Asp-Ile-Ile-Trp, Phe-D-Trp-Leu-Asp-Ile-Ile-Trp,

Val-Tyr-Ile-His-Pro-Phe,

Val-Tyr-Ile-His-Pro,

or the cyclic amino acid sequences

Cyclo- (DTrp-DAsp-Pro-DVal-Leu),

Cyclo- (DGlu-Ala-alloDIle-Leu-DTrp)

on

The present invention furthermore further relates to compounds of the general formula (II)

R ¹ S-CR ² R ³ - (CR ⁴ R ⁵ ) _{n = 1/2} -S-CHR ⁶ -CHR ⁷ -S0 ₂ -NH- (CR ⁸ R ⁹ ) _{m = lf 2} -CO-B (II ) where

R ^{1 represents} a hydrogen atom, a branched or unbranched C 1-6 alkyl radical or a sulfur protecting group,

R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ - R ⁸ and R ^{9 are the} same or different and each represents a hydrogen atom and / or a branched or unbranched Cχ_g alkyl radical,

B for a hydroxyl group, a mercapto group or one

Rest -NHR ^a , wherein

R ^{a is} a hydrogen atom, a branched or straight-chain, cyclic or polycyclic C _] __3 _Q - alkyl, alkenyl, polyalkenyl, alkynyl, Polyalkynyl, aryl, alkylaryl or arylalkyl, which is optionally substituted with hydroxy, oxy, oxo, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde or alkoxy groups having up to 20 carbon atoms and / or optionally is interrupted and / or substituted by one or more heteroatoms from the series 0, N, S, P, As, Se,

their conjugates with substances which selectively accumulate in diseased tissue, a covalent bond being formed between them and, in the case of substances containing carboxyl or amino groups, such as naturally occurring or modified substances

Oligonucleotides in which the abbeu is prevented or made more difficult by naturally occurring nucleases, peptides, proteins, antibodies or their fragments are amidic or, in the case of substances containing hydroxyl groups, such as fatty alcohols, ester-like or, in the case of substances containing aldehyde groups, imidic

and their complexes with radioisotopes of Tc and Re.

The compounds of the general formula (II) according to the invention are prepared by reacting 2-chloroethanesulfonic acid chloride optionally substituted with R ³ and R ⁴ in a manner known per se in an aprotic solvent with addition of a suitable base with compounds of the general formula (III)

H ₂ N- (CR ⁸ R ⁹ ) _{m = 1/2} -CO-B

(III) wherein R ⁸ , R ⁹ , m and B have the meaning given above,

to compounds of the general formula (IV)

R ⁴ HC = CR ⁵ -S0 ₂ -NH-CR ⁶ R ⁷ - (CR ⁸ R ⁹ ) _{m = 1/2} -CO-B

(IV)

wherein R ⁴ - R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , m and B have the meaning given above,

implements.

These reactions are carried out in polar and non-polar, aprotic solvents such as

Dichloromethane, tetrahydrofuran, chloroform, 1,4-dioxane, pyridine, DMF or DMSO at temperatures between -40 ° and 120 ° C with the addition of an auxiliary base to trap the acids released. Such auxiliary bases can, for example, tertiary amines, alkali and

Alkaline earth hydroxides, alkali and alkaline earth carbonates.

The resulting compounds of the general formula (IV) are, if appropriate with the addition of a suitable auxiliary base, such as, for example, a tertiary amine, with compounds of the general formula (V)

R ¹ S-CR ² R ³ - (CR ⁴ R ⁵ ) _{n = 1/2} -SH

(V)

wherein R ¹ , R ² , R ³ , R ⁴ , n and B have the meaning given above,

implements and any protective groups present are split off in a manner known per se.

The invention furthermore relates to kits which are used to produce radiopharmaceuticals, consisting of a compound of the general formula (II) or a conjugate according to the invention containing compounds of the general formula (I and / or II) and substances which accumulate selectively in tissues, a Reducing agents and optionally an auxiliary ligand, which are in the dry state or in solution, as well as instructions for use with a reaction instruction for reacting the described compounds with technetium-99m or Re in the form of a pertechnetate solution or perrhenate solution.

The invention also relates to a radiopharmaceutical composition for the non-invasive in vivo presentation of organs, receptors and receptor-containing tissue and / or of atherosclerotic plaques, which contain a compound of the general formula (I) or a conjugate according to the invention containing compounds of the general formula (I and / or II) and contains substances which accumulate selectively in tissues, optionally with the additives customary in galenicals, the compound being prepared in a kit with technetium-99m or Re in the form of a pertechnetate or perrhenate solution.

In a method for performing a radio-diagnostic examination, the radiopharmaceutical composition is administered to a patient in an amount of 0.1 to 30 mCi, preferably 0.5 to 10 mCi per 70 kg of body weight, and the radiation emitted by the patient is recorded. Surprisingly, many of the chelates synthesized and labeled with technetium-99m or Re show a higher stability than comparable N2S2 and N3S systems which are described in the literature. For example, in the case of a substance according to the invention (Example 2) which was coupled to a partial endothelin sequence, no decomposition products could be observed after 24 hours. It was also possible to determine from competition experiments that the Tc-99m or re-chelators described in this invention complex better than the comparable N2S2, N3S and propylene amine oxime systems. The chelates and chelating agents described in the present invention are thus clearly more suitable for diagnostic and therapeutic purposes than the previously known systems. The mild marking conditions are a particular advantage. Thus, after the protective groups have been split off, the ligands according to the invention and their coupling products can be selectively enriched in diseased tissues

Substances at room temperature and at a physiological pH. The choice of suitable protective groups, which can be split off with different reaction conditions depending on the coupling product, always ensures that undesired side reactions cannot occur during the purification of the coupling products. This ensures that no undesired crosslinking reactions or oxidation of free sulfhydryl groups to disulfides occur under cleaning conditions. Such changes often have an adverse effect on the labeling yield and radiochemical purity and thus also on the background due to non-specifically bound technetium. Sulfur protecting groups are established or split off using methods known to the person skilled in the art. The coupling itself selectively in diseased / 12636 PO7DE96 / 01825

19

Tissue-enriching substances also take place according to methods known per se to the person skilled in the art (for example Fritzberg et al .; J. Nucl.Med. 26, 7 (1987)), for example by reacting electrophilic groups of the complex ligand with nucleophilic centers of the selectively in diseased tissues enriching substances or by reaction of nucleophilic groups of the chelator with electrophilic groups of the substances which selectively accumulate in diseased tissues.

The coupling partners include different biomolecules used. So e.g. Ligands that bind to specific receptors and thus show changes in receptor density, these include Peptides, steroid hormones, growth factors and neurotransmitters. Coupling products with steroid hormone receptor-affine substances enable improved diagnosis of breast and prostate carcinomas (S.J. Brandes and J.A. Katzenellenbogen, Nucl .Med.Biol. 15, 53, 1988). Variously, tumor cells have an altered density of receptors for peptide hormones or growth factors, e.g. the "epidermal growth factor" (EgF). The concentration differences can be used for the selective enrichment of cytostatics in tumor cells (E. Abound-Pirak et al.;

Proc.Natl.Acad.Sei. USA 86, 3778, 1989). Other biomolecules are metabolites that can be introduced into the metabolism of the cells and indicate a changed metabolism; these include e.g. Fatty acids, saccharides, peptides and amino acids. Fatty acids coupled to the less stable N2S2 systems have been described in EP-0200492. Other metabolic products such as saccharides, deoxyglucose, lactate and amino acids (leucine, methyl methionine, glycine) were modified with the help of the PET technique for imaging

Metabolism processes used (R. Weinreich, Swiss Med. 8, 10, 1986). Non-biological substances such as misonidazole and its derivatives, which irreversibly bind to cell components in tissues or tissue parts with a reduced oxygen concentration, can also be used for the specific enrichment of radioactive isotopes and thus for imaging tumors or ischemic regions (ME Shelton, J. Nucl. Med. 30, 351, 1989). Finally, it is also possible to couple the new chelating agents to monoclonal antibodies or their fragments, polysaccharides such as dextrans or starches, bleomycins, hormones, enzymes, polypeptides such as polylysine and nucleotides of the DNA or RNA type. Coupling products of the chelates according to the invention or their complexes with technetium-99m or Re have proven particularly favorable

Endothelines, endothelin derivatives or with partial sequences of the endotheline or their derivatives for the detection of atherosclerotic vascular diseases. These derivatives were applied to WHHL rabbits, which have high LDL concentrations in the blood due to a genetic defect in the LDL receptor and thus have atherosclerotic lesions. About 1 to 6 h after application of the derivatives in WHHL rabbits, a high accumulation in atherosclerotic plaques could be demonstrated. So far, only very late stages of atherogenesis have been diagnosed with invasive procedures. The compounds according to the invention therefore offer the decisive advantage of diagnosing much earlier stages of atherosclerosis using a non-invasive method. Coupling products of the chelates according to the invention or their complexes with technetium-99m or Re with fatty alcohols, fatty alcohol derivatives or with fatty alcohol amines or their derivatives have proven to be favorable for the detection of atherosclerotic vascular diseases. These derivatives were applied to WHHL rabbits by a genetic defect of the LDL receptor have high LDL concentrations in the blood and thus have atherosclerotic lesions. About 1 to 6 h after application of the derivatives in WHHL rabbits, a high accumulation in atherosclerotic plaques could be demonstrated. So far, only very late stages of atherogenesis have been diagnosed with invasive procedures. The compounds according to the invention therefore offer the decisive advantage of diagnosing much earlier stages of atherosclerosis using a non-invasive method.

It is immaterial whether the described chelating agents are labeled with technetium-99m before or after coupling to the selectively enriching molecule. For coupling to the selectively enriching molecule after complexation, however, it is a prerequisite that the reaction of the radioactive complex with the enriching compound proceeds quickly, under mild conditions and almost quantitatively, so that subsequent purification is not necessary.

The pharmaceutical compositions according to the invention are prepared in a manner known per se by adding the complexing agents according to the invention with the addition of a reducing agent, preferably tin (II) salts such as chloride, pyrophosphate or tartrate, and optionally with the addition of galenics usual additives - dissolves in aqueous medium and then sterile filtered. Suitable additives are, for example, physiologically harmless buffers (eg tromethamine), small additions of electrolytes (eg sodium chloride), stabilizers (eg gluconate, phosphates or phosphonates). The pharmaceutical composition according to the invention is in the form of a solution or in lyophilized form and is shortly before application with a solution of Tc-99m pertechnetate, eluted from commercially available Mo / Tc generators, or a perrhenate solution.

When used in nuclear medicine in vivo, the pharmaceutical compositions according to the invention are dosed in amounts of 1x10 "5 to 5χio ⁴ nmol / kg body weight, preferably in amounts between lxlO ^-3 to 5xl0 ² nmol / kg body weight. Starting from an average body weight of 70 kg the amount of radioactivity for diagnostic applications is between 0.05 to 50 mCi, preferably 5 to 30 mCi per 70 kg application, for therapeutic applications between 5 and 500 mCi, preferably 10 to 350 mCi. The application is normally carried out by intravenous, intraarterial, peritoneal or intertumor injection of 0.1 to 2 ml of a solution of the agents according to the invention, intravenous application is preferred.

The following examples serve to explain the subject of the invention in more detail.

example 1

N-vinylsulfonylglycine methyl ester (1) An ice-cooled solution of 8.91 g (100 mmol) of the glycine methyl ester and 17.9 g of chloroethanesulfonyl chloride (110 mmol) in 100 ml of dichloromethane is slowly added with dry pyridine (400 mmol) while cooling with ice. The mixture is allowed to warm to RT and, after the reaction has ended, 200 ml of dilute HCl are added and the dichloromethane phase is separated off. The aqueous phase is extracted several times with dichloromethane, washed with water, dried, concentrated and chromatographed (silica gel CH2CI2) - 12.9 g of a slowly crystallizing oil remain. Yield: 72% Analysis: calc. : C 33.52 H 5.06 N 7.82 0 35.72 S 17.90 Found: C 33.24 H 5.23 N 7.66 S 17.61

N- (5-hydroxy-3-thiapentylsulfonvl) -αlycin methyl ester (2) To a stirred solution of 7.81 g mercaptoethanol (100 mmol) and 150 mg Triton-B solution are added

Cooling 1.79 g of vinylsulfonic acid 1 (10 mmol) were added and the mixture was stirred at RT for 20 h with exclusion of oxygen. Then water is added and the mixture is extracted several times with ethyl acetate. The combined organic extracts are washed with bicarbonate solution

Dried sodium sulfate and concentrated. The residue is chromatographed (silica gel, EtOAc). Yield: 63% Analysis: Found: C 32.67 H 5.88 N 5.44 O 31.09 S 24.92 Found: C 32.59 H 5.75 N 5.41 S 24.86

N- (5-chloro-3-thiapentylsulfonyl) σlycine methyl ester (3) A solution of 2.57 g of glycine derviate 2 (10 mmol) in 50 ml of anhydrous carbon tetrachloride is mixed with 3.14 g of powdered triphenylphosphine (12 mmol) under a nitrogen atmosphere. added and heated under reflux. After cooling, it is diluted with 50 ml of hexane and kept at -20 ° C for some time. The precipitate is suctioned off and the procedure is repeated as above until none

Precipitation fails more. It is then dried and concentrated. Yield: 62% Analysis: calc. : C 30.49 H 5.12 N 5.08 0 23.21 S 23.26 Found: C 30.16 H 5.56 N 5.33 S 23.24 N- (5-thiouronyl-3-thiapentylsulfonyl) σlycin methyl ester 141

The solution of 5.51 g of 2. (20 mmol) in ethanol is added dropwise to a solution of 1.52 g of thiourea in ethanol and the mixture is then boiled under reflux for 2 h. After the solvent has been stripped off, a crystalline residue remains which is recrystallized from ethanol. Yield: 57% Analysis:

Ber. : C 27.31 H 5.16 N 11.94 0 18.19 S 27.34 Found: C 27.11 H 5.52 N 11.54 S 27.80

N- (5-mercapto-3-thiapentylsulfonyl) glycine (5) 3.52 g (10 mmol) 4. are stirred under protective gas in aqueous methanolic potassium hydroxide solution at 50 ° C. for 3 hours. After cooling, it is diluted with 400 ml of water and the undissolved solution is filtered off. The filtrate is acidified with HCl, extracted with dichloromethane, dried, concentrated and recrystallized. Yield: 64% Analysis:

Calc .: C 27.79 H 5.05 N 5.40 O 24.68 S 37.09 Found: C 28.17 H 5.35 N 5.48 S 36.61

N- (5-mercapto-3-thiapentylsulfonyl) glycine, technetium

99m complex

10 mg of compound 5_ are dissolved in 1.0 ml of ethanol. 50 ul of this ligand solution are mixed with 100 ul ethanol, 150 ul phosphate buffer pH 8.5, 50 ul of a deoxygenated aqueous citrate solution (50 mg / ml), 2.5 ul of a deoxygenated tin (II) chloride solution (5 mg / ml 0.1 N HCl) and 100 μl of a pertechnetate solution (400-1000 μCi) are added. After an incubation time of 10 min, the reaction mixture is examined for the purity of the Tc complex formed by means of HPLC: LiChrospher RP-18 Column, 5µ, 125 x 4.6mm; Gradient elution from 100% A to 100% B within 15 min (eluent A: acetonitrile / sodium phosphate 5 mM, pH 2.0 (10/90); eluent B: acetonitrile / sodium phosphate 5 mM, pH 2.0 (75/25); 1 ml / min. The radiochemical purity is> 92%.

Example 2

N- r (5-triphenylmethγlmercapto) -3-thiapentylsulfonyll - glycine (6)

The solution of 2.79 g of triphenylchloromethane in DMF is slowly added dropwise to a solution of 2.59 g of the glycerol derivative 5_ (10 mmol) and triethylamine (10 mmol) in DMF and the mixture is stirred at RT for 12 hours. Then it is mixed with water, weakly acidified with dilute hydrochloric acid and extracted with dichloromethane. The organic phase is dried and concentrated. The remaining residue is chromatographed (silica gel, CH ₂ Cl ₂ / MeOH). Yield: 57% Analysis:

Ber. : C 59.86 H 5.43 N 2.79 0 12.76 S 19.18

Found: C 59.46 H 5.66 N 2.44 S 19.11

HOOC-Trp-Ilft-T ^] fi-Asp-Leu-D-Trp-Phe- {N- \ ⁽ 5-

Triphenylmethylmercapto) -3-thiapentvlsulfonyll Glv} LH

To a solution of 502 mg of the acid ß_ (1 mmol), 280 μl of triethylamine and 115 mg of N-hydroxysuccinimide (1.0 mmol) in 10 ml of anhydrous dimethylformamide, 211 mg of EDC (1.1 mmol ) added dropwise in 5 ml of anhydrous dimethylformamide and stirred at 0 ° C. for 2 hours. A solution of 992 mg of H ₂ N-Phe D-Trp-Leu-Asp-Ile-Ile-Trp-COOH (1.0 mmol) in DMF is then added dropwise within 30 minutes. The mixture is first stirred at 0 ° C. for a further 2 hours and at 12 hours Room temperature stirred. The product is filtered off from the urea and the filtrate is evaporated in vacuo and taken up in dichloromethane. After renewed filtration, the mixture is washed twice with 0.5 N HCl and saturated sodium hydrogen carbonate solution, dried over magnesium sulfate and the solvent is stripped off. The residue is crystallized by trituration with diethyl ether. Yield: 33% Analysis: calc. : C 63.48 H 6.42 N 9.49 O 14.09 S 6.52 Found: C 63.09 H 6.66 N 9.37 S 6.46

HOOC-Trp-Tle-Ile-Asp-Leu-D-Trp-Phe-fN- (5-mercapto-3-thiabutylsπ ifonyl) Glγ1 Lül 1.48 g of peptide 1 (1 mmol) is 45 minutes at 0 ° C With

20 ml of anhydrous HF in the presence of 5 ml of anisole and

3.5 ml of diethyl sulfide treated. After the acid has been evaporated off, the remaining residue is taken up in 5% acetic acid, washed several times with diethyl ether and lyophilized. Chromatographic purification via Sephadex

G-10 with 0.2 M acetic acid gives 470 mg of an oil.

Yield: 38%

Analysis:

Ber. : C 57.45 H 6.54 N 11.36 0 16.86 S 7.80 Found: C 57.34 H 6.43 N 11.40 S 7.92

HOOC-Trp-ne-Ile-Asp-T.eu-D-Trp-Phθ-TN- (5-mercapto-3-thiapentylsulfonyDGlyl. Technetium-99m complex

10 mg of compound £ are dissolved in 1.0 ml of ethanol. 50 μl of this ligand solution are mixed with 100 μl ethanol,

150 ul phosphate buffer pH 8, 5, 50 ul of a deoxygenated aqueous citrate solution (50 mg / ml), 2.5 ul of a deoxygenated tin (II) chloride solution (5 mg / ml 0.1 N HCl) and 100 ul of a pertechnetate Solution (400-1000 μCi) added. The reaction mixture is after a

Incubation time of 10 min using HPLC for purity of the Tc complex formed: LiChrospher RP-1. Column, 5 µ, 125 x 4.6 mm; Gradient elution from 100% A to 100% B within 15 min (eluent A: acetonitrile / sodium phosphate 5 mM, pH 2.0 (10/90); eluent B: acetonitrile / sodium phosphate 5 mM, pH 2.0 (75/25); 1 ml / min. The radiochemical purity is> 96%.

Claims

claims

1. Compounds of the general formula (I)

M - L (I)

wherein

M is a radioisotope of Tc or Re and L is a ligand of the general formula (II)

RIS-CR ² R ³ - (CR ⁴ R ⁵ ) _{n = 1/2} -S-CHR ⁶ -CHR ⁷ -S0 ₂ -NH- (CR ⁸ R ⁹ ) _{m = 1 # 2} -CO-B

(II) means where

R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ <R ⁸ and R ^{9 are the} same or different and each represents a hydrogen atom and / or a branched or unbranched C _] __g alkyl radical,

B for a hydroxyl group, a mercapto group or a radical -NHR ^a , wherein

R ^{a is} a hydrogen atom, a branched or straight-chain, cyclic or polycyclic C _x _3Q-alkyl, alkenyl, polyalkenyl, alkynyl,

Polyalkynyl, aryl, alkylaryl or arylalkyl radical, optionally with hydroxy, oxy, oxo, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde or alkoxy groups with up to 20 carbon atoms 97/12636 PO7DE96 / 01825

29 is substituted and / or optionally interrupted and / or substituted by one or more heteroatoms from the series 0, N, S, P, As, Se.

2. Compounds according to claim 1, characterized in that n and m each represent 1.

3. Compounds according to claim 1 or 2, characterized in that R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and R ^{9 represent} hydrogen atoms.

4th Ligands of the general formula (I I)

R ¹ S-CR ² R ³ - ⁽ CR ⁴ R ⁵⁾ _{n = lf 2} - ^S - ^CHR6 - ^CHR7 - ^SO 2 - ^NH - ^{CR8R9 > Π. = 1, 2 ^{- C} ° - ^B

(I I)

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , n, m and B each have the meaning given in claim 1.

5. Ligand according to claim 4, characterized in that n and m each represent 1.

6. Ligands according to claim 5 or 6, characterized in that R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ^{9 represent} hydrogen atoms.

7. Conjugates containing a compound of the general formula (I and / or II) and substances which accumulate selectively in diseased tissue, a covalent bond being formed between them and, in the case of substances containing carboxyl or amino groups, such as naturally occurring or modified oligonucleotides where the Abbeu is prevented or made more difficult by naturally occurring nucleases, peptides, proteins, antibodies or their fragments are amidic or, in the case of substances containing hydroxyl groups, such as fatty alcohols, ester-like or in the case of

Substances containing aldehyde groups are imidic.

8. Conjugates according to claim 7, characterized in that the accumulating in diseased tissue

Substances mean peptides such as endotheline, partial sequences of endothelin, endothelin analogs, endothelin derivatives, endothelin antagonists or angiotensins, partial sequences of angiotensins, angiotensin analogs, angiotensin derivatives and angiotensin antagonists, and chemotactic peptides.

9. Conjugates according to claim 7, characterized in that the peptides have the following sequences or parts thereof

I 1

Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr

Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Ser-Cys-Ser-Ser-Trp-Leu-Asp-Lys-Glu-Cys-Val-Tyr-

Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Thr-Cys-Phe-Thr-Tyr-Lys-Asp-Lys-Glu-Cys-Val-Tyr- li Phe-Cys-His-Leu-Asp-Ile-Ile-Trp, Cys-Ser-Ala-Ser-Ser-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-

I.

Phe-Cys-His-Leu-Asp-Ile-Ile-Trp,

Cys-Ser-Cys-Lys-Asp-Met-Thr-Asp-Lys-Glu-Cys-Leu-Asn-

Phe-Cys-His-Gln-Asp-Val-Ile-Trp,

Ala-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr- Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,

Ala-Ser-Ala-Ser-Ser-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr- Phe-Ala-His-Leu-Asp-Ile-Ile-Trp,

I 1

Cys-Val-Tyr-Phe-Cys-His-Gin-Asp-Val-Ile-Trp,

N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His ^■ Gln-Asp-Val-Ile-Trp,

Asp-Arg-Va1-Tyr-Ile-His-Pro-Phe-His-Leu,

Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe,

Arg-Val-Tyr-Ile-His-Pro-Phe, Arg -Val - Tyr- I le -His - Pro - Phe -His -Leu,

Sar- Arg- Val -Tyr- Val -His - Pro -Ala,

For-Met - Leu-Phe,

For-Met - Leu- Phe -Lys,

the partial sequences

His-Leu-Asp-Ile-Ile-Trp,

D-Trp-Leu-Asp-Ile-Ile-Trp,

Phe-D-Trp-Leu-Asp-Ile-Ile-Trp,

Val-Tyr-Ile-His-Pro-Phe,

Val-Tyr-Ile-His-Pro,

or the cyclic amino acid sequences

Cyclo- (DTrp-DAsp-Pro-DVal-Leu),

Cyclo- (DGlu-Ala-alloDIle-Leu-DTrp)

exhibit.

10. A process for the preparation of a compound of general formula (I), characterized in that technetium-99m or Re in the form of pertechnetate or perrhenate in the presence of a reducing agent and optionally an auxiliary ligand with a compound of general formula (II) R ¹ S-CR ² R ³ - (CR ⁴ R ⁵ ) _{n = 1 # 2} -S-CHR ⁶ -CHR ⁷ -S0 ₂ -NH- (CR ⁸ R ⁹ ) _{m = 1 # 2} -CO-B

(II)

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , n, m and B have the meaning given in claim 1,

implements.

11. A process for the preparation of ligands of the general formula (II), characterized in that 2-chloroethanesulphonyl chloride optionally substituted with R ⁵ and R ⁶ in a manner known per se with the addition of a suitable base with compounds of the general formula ( III)

H ₂ N- (CR ⁸ R ⁹ ) _{m = 1 # 2} -CO-B

(III)

wherein R ⁸ , R ⁹ , m and B have the meaning given in claim 1,

at temperatures from -20 ° to 180 ° C to compounds of the general formula (IV)

R ⁴ HC = CR ⁵ -S0 ₂ -NH-CR ⁶ R ⁷ - (CR ⁸ R ⁹ ) _{m = 1/2} -CO-B

(IV)

wherein R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , m and B have the meaning given in claim 1,

implements and these compounds of the general formula (IV) optionally with the addition of a suitable auxiliary base at temperatures from -20 ° C. to 180 ° C. in a manner known per se with compounds of the general formula (V)

R ¹ S-CR ² R ³ - (CR ⁴ R ⁵ ) _{n = 1/2} -SH

(V)

wherein R ¹ , R ² , R ³ , R ⁴ , n and B have the meaning given in claim 1,

implements

and any protective groups present are split off in a manner known per se.

12. Kit for the production of radiopharmaceuticals, consisting of a compound of general formula (II) according to one of claims 4 to 6 or a conjugate according to one of claims 7 to 9 and a reducing agent and optionally an auxiliary ligand, which are in the dry state or in solution are available, as well as instructions for use with a reaction instruction for the implementation of the described compounds with Technetium-99m or Re in the form of a pertechnetate solution or perrhenate solution.

13. Radiopharmaceutical composition for the non-invasive in vivo presentation of organs, receptors and receptor-containing tissue and / or of atherosclerotic plaques, characterized in that it contains a compound according to one of claims 1 to 3 or a conjugate according to claims 7 to 9 and optionally with the additives customary in galenics, the compound being prepared in a kit according to claim 12 with technetium-99m or Re in the form of a pertechnetate or perrhenate solution.

14. A method for radiodiagnostic examination, characterized in that a radiopharmaceutical composition according to claim 13 in an amount of 0.1 to 30 mCi, preferably 0.5 to 10 mCi per 70 kg body weight is administered to a patient and the radiation emitted by the patient is recorded becomes.