DE4337600A1 - N-alkyl peptide chelating agents, their metal complexes with radionuclides, processes for their preparation and radiopharmaceutical compositions containing these compounds - Google Patents

Description

The invention relates to N-alkyl peptide chelating agents, their metal complexes with radionuclides, process to their preparation and containing these compounds radiopharmaceutical compositions.

The invention further relates to radiopharmaceuticals which contain these chelates in a form complexed with metals ten, whose diagnostic kits, in which the chelate in non-complex form, and either by adding technetium or rhenium ions is xiert and also on the use of such preparations for diagnostic and therapeutic purposes.

Radioactive substances are mainly used in the medical diagnostics applied to deformities and Functional changes of internal organs or changes patho establish logical processes in the body. For this Application is given to the patient a formulation enough, e.g. B. in the form of a solution for injection, which the contains radioactive substance. With suitable detectors (e.g. a gamma camera) by recording the emitted radiation images of organs, pathological Maintain processes and their changes in the body become; with particularly high accumulations in organs or vessels they serve as therapeutic agents.

In the past few years, the demand for specialty has grown fishing, radioactive labeled chemical compounds, the so-called carrier molecules (e.g. steroids, antibodies per, lipids, proteohormones etc.) can be coupled and act on the principle of "drug targeting". Of of particular interest for tumor diagnosis and therapy as well as for receptor display (e.g. steroid conjugates,  Antibodies, etc.), are such substances that are empowered are to pass the cell wall and the blood-brain barrier and substances for checking organ functions, e.g. B. before and after transplants and Finding vascular lesions. Aiming for a higher one Selectivity of the radiopharmaceutical and one with it associated significant enrichment of a radionuclide To get in the target organ, complexing agents are increasing developed and used for a variety of radionuclides tissue or metabolism-specific carrier molecules coupled. For therapy, you are looking for ways to use a radioactive isotope of rhenium.

The most commonly used radionuclide is technetium-99m, which due to its favorable physical properties (no corpuscular radiation, favorable physical half-life, 140 KeV γ radiation) and the associated low radiation exposure is particularly suitable as an isotope for in-vivo diagnostics. Techne tium-99m can be easily obtained from nuclide generators as [ ^99m Tc] pertechnetate. To obtain techne tium-99m chelates, the pertechnetate is converted into a lower oxidation state by suitable reducing agents (e.g. SnCl₂, S₂O₄ ^2- , etc.), in which the technetium-99m forms complexes with chelating agents or proteins. Special procedures have been developed and described for labeling potential chelating agents with technetium-99m and for producing kits for routine clinical needs. Proteins can be obtained directly via donor groups (amino, amide, thiol, etc.) of the protein (J. Nucl. Med. 1986, 27, 685) or by introducing complexing agents (US Pat. Nos. 4,479,930 and Fritzberg, AR et al, J. Nucl. Med. 1986, 27, 957) with technetium-99m.

Cyclic amines also became complex ner developed (Troutner, D.E. et al; J. Nucl. Med. 1980, 21, 443 and Mäcke, H .; DE-39 11 816), Cyclame (Ketring, A.R .; Troutner, D.E. et al; J. Nucl. Med., 1980, 21, 443-448, Int. J. Nucl. Med. Biol. 1984, 11, 113, Volkert et al, Applied Radiat. Isot., 1982, 33, 891-896), N₂O₂ systems (Pillai, M.R.A .; Troutner, D.E. et al; Inorg. Chem. 1990, 29, 1850) and also N₂S₂ systems (Bormans, G. et al; Nucl. Med. Biol. 1990, 17, 499) and N₃S systems (Fritzburg, A .; EP-A-01 73 424 and EP-A-02 50 013).

However, all of these complexing agents have significant aftermath parts. So are for marking the cyclame as well N₂O₂ systems known from the literature (Pillai, M.R.A. et al; Inorg. Chem. 1990, 29, 1850-1856) pH values of 10-13 required. Furthermore, these systems do not have reactive groups that link to biomolecules or enable proteohormones. N₂S₂ and N₃S systems have sufficient stability in individual cases but are only for simple excretion studies such as urine output, EP-A-02 50 013, suitable net. Efforts to produce the literary MAG₃ (EP-A-02 50 013), which a Erhit for marking zen to 100 ° C (Bannister, K.M. et al; J. Nucl. Med. 1990, 31, 1568-1573) are to be simplified in Gange (WO-91-16 076), but to date without a clinical Access remained.

For N₂S₂ complexes (Bormans, G. et al; Nucl. Med. Biol. 1990, 17, 499) is the shelf life between preparation and administration less than 1 hour, meaning one sensitive disadvantage. So here too - so far of no clinical relevance - efforts underway to address deficiencies resolve (Merryn, W. et al, Applied. Radiat. Isot. Vol. 42 (7), 607-612).  

The complexing agents that have become known have ever because a uniform structure and are therefore also diagnostically applicable for one purpose only. The molecule Variations are on substituents on the backbone limited and therefore do not allow a wide range of medicine application as is possible with a complex variation would be. They are therefore expensive to manufacture and scientific penetration of the individual com plexe.

The invention has for its object to provide short-chain N-alkyl peptide chelating agents which complex with technetium and rhenium in a wide pH range at room temperature, both ionic and non-ionic complexes being formed and thus providing compounds which have the disadvantages known ^99m Tc and ^180,188 Re complexes do not have.

At the same time, the requirements for Use of these compounds in humans With regard to radiation dose, stability and solubility be fulfilled; it must be as simple as possible at the same time Methods for their representation are created and a Kit formulation of this invention Compounds / conjugates and their metal complexes for their clinical application are provided.

This object is achieved in that Compounds of the general formula I

R³-S-CH₂-CO-NR¹- [CH₂-CO-NH] _n -CH₂-CO-R² (I)

wherein
n represents a number 0, 1 or 2,
R¹ is a straight-chain or branched alkyl radical having 1 to 20 carbon atoms, which is optionally interrupted or substituted by one to three oxygen atoms, and which optionally carries a terminal - COOH, -OH or -NH₂ group, which optionally with glycolic acid or glycolic acid esters or ether is esterified or etherified,
the esters or ethers being formed with carboxylic acids or alcohols having up to 18 carbon atoms,
or represents a phenyl or cyclohexyl radical which is optionally in the 4-position with a COOH, NH₂ or OH group which is optionally esterified, etherified or amidated with carboxylic acids or alcohols having up to 6 carbon atoms or is substituted with a halogen atom,
R² represents a halogen atom, a halogenomethyl, methylcarboxyl, trifluoromethylcarboxyl, an NH₂ or an OH group,
where the carboxyl group formed in the case of R 2 = OH either directly or after esterification with an α, ω-hydroxycarboxylic acid having up to 8 carbon atoms via its terminal carboxyl group with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, one Cholecystokinin, a peptide, a protein, a proteohormone, an aminosugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment is esterified or amidated,
a radical of the general formula II

or the general formula IIa

is where
R⁴ is a methylene, propenylene amino, propynylene amino, methylene amino or methyleneoxy group and
R⁸ means a hydrogen atom or a methyl group,
a radical of the general formula III

or the general formula IIIa

represents what
R⁵ is a -NH-, -NH-CO-N <, -NH-CO-NH- or methyleneoxy group,
R⁶ is a hydrogen atom, a halogen atom or a methyl group and
R⁷ represents a hydrogen atom or a straight-chain or branched alkyl radical having up to 6 carbon atoms,
R³ represents a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethylacetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or an easily removable sulfur protecting group
and their salts are provided with pharmaceutically acceptable acids or bases.

Surprisingly, a class of compounds was found which not only avoids the disadvantages mentioned, but as a ^99m Tc ⁺⁵ complex has a transition from the N₃S → N₂SO → S₂O₂ → N₂S₂-N-alkyl-peptide complex system and therefore has a wide range as a com plexer can be used.

Formula IV shows an N₃S-N-alkyl-peptide complex in the ^99m Tc core (neutral)

R³S-CH₂-CO-NR¹ [CH₂-CO-NH] ₂-CH₂-CO-R²,

the formula V from an N₂OS-N-alkyl-peptide complex in the ^99m Tc core (neutral)

R³S-CH₂-CO-NR¹ [CH²-CO-NH] ₁-CH₂-COOH,

the formula VI from an O₂S₂-N-alkyl-peptide complex in the ^99m Tc core (with negative charge)

R³S-CH₂-CO-NR¹ [CH₂-CO-NH] ₀-CH₂-COOH,

and formula VII finally shows an N₂S₂-N-alkyl-peptide complex in ^99m Tc core (with positive charge)

R³S-CH₂-CO-NR¹ [CH₂-CO-NH] ₀-CH₂-CO-R².

The complexes that result from the molecular variation show a surprising breadth and agreement with regard to their biological behavior. Thus, compounds of this type, coupled to a bioligand, can be used for receptor display, which has not previously been described for ^99m Tc compounds and are outstandingly suitable for displaying plaques in vascular lesions and for measuring kidney function, the compounds each being coupled to bioactive ligands could be.

Preferred are compounds in which R¹ represents a -CH₂- (CH₂) _m -CH₃ group with m = 1-14
or R1 is phenyl, p-phenylamine, cyclohexylamine, p-hydroxyphenyl, 4-hydroxycyclohexyl, p-halophenyl or 4-halocyclohexyl.

Compounds of the general are also preferred Formula I, wherein

R¹ is - (CH₂) _q -OOC-CH₂-OOC- (CH₂) _r -CH₃,

where q and r each represent integers from 1 to 16.

Compounds of the general are particularly preferred Formula I, wherein R¹ is a straight chain or branched Alkyl radical with 1 to 6 carbon atoms or a P- Is bromophenyl.

Furthermore, compounds of general formula I are before moves in which R² is a radical of the general formula II

or the general formula IIa

is where
R⁴ is a methylene, propenylamino, propinylamino, methylenamino or methyleneoxy group and
R⁸ means a hydrogen atom or a methyl group.

Of these steroids, 17α-ethynyl estradiols or whose 3-methyl ether is preferred.

A 17α-ethynyl group of estradiol can also be coupled be with an ethene. The order of ethins and Ethene groups are interchangeable; also doubling the Ethine or ethene group is appropriate. The important thing is Minimum distance between chelator and estradiol molecule, preferably a rigid ethyne or ethene group, so that the side chain cannot fold.

I binds in this preferred 17α-position tion of the estradiol scaffold, is in the preserved Che lat complex bioligand compounds none or only one expected a slight decrease in biological activity how it z. B. in Epperly, M.W. et al [J. Steriod biochem. Molec. Biol. Vol. 39, No. 5A, 729-734, 1991].

The outstanding importance of these estrogen-chelate complexes lies in their peculiarity of penetrating the cell wall and attaching to the estrogen receptor. This applies in particular to complexing agents according to the general formula I, in which n = 2. At ^99m Tc ⁺⁵ these N₃S complexes are neutral as a result of the N¹-alkylation in the Tc core (cf. Formula IV). While charged complexes are unable to penetrate the cell wall, e.g. B. N₃S complex as a steroid 17α-ethynyl chelate, of the thioacetyl-glycyl-glycyl-glycyl-O-ester type (own experiments), penetrate the N¹-alkylated complexes according to the invention (N¹ = methyl) and enrich the cell wall iv injection in the uterus. The enrichment, expressed as a quotient between blood / uterus, is 0.2 and can be varied depending on the N 1 -alkyl chain length or substituents. This enables a mamma-ca early detection using the SPECT method. The compound according to Example 10 is particularly preferred here.

Also preferred are compounds of general formula I in which R² is a radical of
general formula III

or the general formula IIIa

is where
R⁵ is an -NH-, -NH-CO-N <, -NH-CO-NH- or methylene oxy group,
R⁶ is a hydrogen atom, a halogen atom or a methyl group and
R⁷ represent a straight-chain or branched alkyl radical having up to 6 carbon atoms.

These compounds of general formulas III and IIIa are ergoline derivatives.

Of these ergolines, the 8α-amino-6-methyl-ergoline is optionally with alkyl substituents in the 2- and / or 6-position with a chain length of C₁ in the 2-position or C _1-3 in position 6 and / or one Methyl or halogen substituents in position 2 are preferred. The substituent in 8-position of the ergoline can, in addition to αNH₂, also be β-CH₂O- or αNH-CO- N <or αNH-CO-NH-.

The compounds of the general formula according to the invention I have a residue R³ at the terminal sulfur atom. This R³ represents a sulfur protection group, which during the synthesis of the verbin of general formula I is required. A such a protective group must be easily removable. Suitable Rest R³ are, for example, acetyl, benzoyl, p-methoxy benzyl, acetamidomethyl, benzamidomethyl, trime thylacetamidomethyl, hydroxyacetyl, ethoxyethyl or Trityl groups. Benzyol is particularly preferred group.

The present invention also relates to Metal chelate complexes of radioactive metal ions with the compounds of the general formula I according to the invention, wherein R¹, R² and R³ have the meaning given above.  

Suitable radioactive metal ions are, for example Ions of the radioisotopes of the elements Tc, Re, Cu, Ga, Gd, Y and in. The selection of the radionuclide depends on the desired type of application of the invention Metal chelate complexes of the general formula I.

Thereby be used for radio diagnostics or radiotherapy different radionuclides are used.

Radioactive metal ions of Iso are preferred tope of the elements Tc and Re.

The radioisotope technetium-99m is particularly preferred.

Another object of the present invention are Conjugates containing compounds of the general formula I or metal chelate complexes of radioactive metal ions Elements Tc, Re, Cu, Ga, Gd, Y and In with connections of general formula I and selectively in diseased Tissues or substances accumulating in tumors there is a covalent bond between them and this in the case of carboxy or amino groups Substances such as peptides, proteins, antibodies or their fragments, amidic or in the case of hydroxy group pen-containing substances such as fatty alcohols, ester-like or in the case of substances containing aldehyde groups is imidic.

Conjugates with diseased tissue are preferred enriching peptides such as endotheline, partial sequences of Endothelins, endothelin analogs, endothelin derivatives or endothelin antagonists.

Such conjugates with the are particularly preferred Compounds of general formula I and themselves selective  peptides accumulating in diseased tissue, the Peptides the following sequences

exhibit.

Another object of the present invention is a process for the preparation of the invention Compounds of the general formula I

R³-S-CH₂-CO-NR¹- [CH₂-CO-NH] _n -CH₂-CO-R² (I)

wherein
n represents a number 0, 1 or 2,
R¹ is a straight-chain or branched alkyl radical having 1 to 20 carbon atoms, which is optionally interrupted or substituted by one to three oxygen atoms, and which optionally carries a terminal -COOH, -OH or -NH₂ group, which optionally with glycolic acid or glycolic acid esters or ether is esterified or etherified,
the esters or ethers being formed with carboxylic acids or alcohols having up to 18 carbon atoms,
or represents a phenyl or cyclohexyl radical, which cher optionally in the 4-position with a COOH, NH₂ or OH group, which is optionally esterified, etherified or amidated with carboxylic acids or alcohols having up to 6 carbon atoms or a halogen atom is substituted,
R² represents a halogen atom, a halogenomethyl, methyl carboxyl, trifluoromethylcarboxyl group, an NH₂ or an OH group,
where the carboxyl group formed in the case of R 2 = OH either directly or after esterification with an α, ω-hydroxycarboxylic acid having up to 8 carbon atoms via its terminal carboxyl group with a biomolecule, a steroid, an ergoline derivative, a benzodiazine derivative, a cholecystokinin, a peptide, a protein, a proteohormone, an aminosugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment is esterified or amidated,
a radical of the general formula II

or the general formula IIa

is where
R⁴ is a methylene, propenylene amino, propynylene amino, methylene amino or methyleneoxy group and
R⁸ represents a hydrogen atom or a methyl group,
a radical of the general formula III

or the general formula IIIa

represents what
R⁵ is an -NH-, -NH-CO-N <, -NH-CO-NH- or methylene oxy group,
R⁶ is a hydrogen atom, a halogen atom or a methyl group and
R⁷ represents a hydrogen atom or a straight-chain or branched alkyl radical having up to 6 carbon atoms,
R³ represents a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethylacetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or a readily removable sulfur protective group
and their salts with pharmaceutically acceptable acids or bases
which is characterized in that one

• a) a diketopiperazine derivative of glycine or
• b) glycine

with a halocarboxylic acid halide and then with an alkylamine or arylamine of the general Formula VI

R¹-NH₂ (VI)

where R¹ has the meaning given above, reacted again with a halocarboxylic acid halide and then with a compound of general Formula IV

R³-SH (IV)

where R³ has the meaning given above,
implements
or a compound of the general formula V

R¹-NH-CH₂-CO-R² (V)

where R² has the meaning given above, with a Halocarboxylic acid halide and then with a Alkylamine or arylamine of the general formula VI

R¹-NH₂ (VI)

where R¹ has the meaning given above, reacted again with a halocarboxylic acid halide and then with a compound of the general formula IV

R³-SH (IV),

where R³ has the meaning given above and these compounds optionally with a pharma acceptable acid or base in their salt leads.

The compounds of the general formula according to the invention I can also be prepared in such a way that one starts from a compound of the general invention Formula I, where n is 0 or 1, this with a verb implementation, which has terminal groups, and thereby after coupling to the above connection forms a radical R². Thereby according to the invention  Compounds of general formula I formed in which n is 1 or 2. This means that part of the chain of Compounds of the general formula I according to the invention provided by the attached rest R² becomes.

The production of the metal chelate according to the invention plexes of radioactive metal ions with the invention Compounds of general formula I is another Subject of the present invention.

The reaction of the compounds of the general formula I if necessary under previous or simultaneous The rest of R³ is split off in a manner known per se Wise.

The production of metal chelate complexes radioactive Metal ions of elements Tc and Re with compounds of general formula I takes place in that technetium 99m or Re in the form of pertechnetate or perrhenate in Presence of a reducing agent and optionally of an auxiliary ligand with a compound of the general Formula I.

R³-S-CH₂-CO-NR¹- [CH₂-CO-NH] _n -CH₂-CO-R² (I)

wherein R¹, R² and R³ have the meaning given above, implements.

The is of particular inventive importance surprising variability of the compounds and their Complexes. For connections with n = 2 according to the general NEN formula I gives an N₃S system. If one goes from n = 2 to n = 1, so there can be an N₂SO-N-alkyl system form with a surprisingly high complex stability. Finally, at n = 0, the N₁S₁-N-alkyl  System when complexing a chelate complex that both shows an N₂S₂-N-alkyl system or as another Dimeres is an O₂S₂-N-alkyl complex, without a chemical covalent bridge function the complex connects.

The new compounds have because of their chemical Variability and its variants in complex formation also an extraordinary range of clinical applications dung.

To date, there is no method that - in vivo - that Plaque formation as the onset of atherosclerosis figuratively. Atherosclerosis is a folk disease and a precursor to a coronary heart crane kung, the consequences of which about 40% of the population of Industrialized countries are dying. It is therefore special Value, with a simple means to a pictorial Representation of vascular lesions. So they can N-alkyl chelates according to the invention for early detection atherosclerotic vascular diseases can be used.

The quotient obtained in the animal model (rabbits of the species WHHL with artificially generated atherosclerotic vascular changes: WHHL rabbits have high LDL levels in the blood due to a missing or defective LDL receptor and therefore develop atherosclerotic vascular changes) after iv administration of compounds according to the general formula I , where R² is an endothelin, a partial sequence of endothelin, an endothelin analogue, an endothelin derivative or an endothelin antagonist, from activity in the plaque and undamaged vessel, was 1: 5 (cf. FIGS. 1 and 2) .

It was highly surprising that despite the Lipho philie of the complex, as a result of the R¹ alkyl chain  a chain length of C₆ already after 3 hours Elimination so far. The lesions in the area the aorta are surprisingly well represented autoradiographically adjustable. Here lies one due to the fat similarity of the Molecules given affinity for the plaques or their Receptors before that is so high that despite falling Concentration of the compound in the blood - as a result of Elimination via the liver - adherence to the plaques preserved.

Affinity for plaques in atherosclerotic changes of the vessels can be varied by varying the R¹- Influence alkyl group. So can be about the peculiarity of the rest in R¹ control lipophilicity, and at the same time, R² offers the as a free carboxyl group necessary prerequisite for good water solubility. Instead of R² as a free carboxylic acid, one can also Amidation of this carboxyl group with an amino carbon hydrogen contribute to water solubility.

It is known that proteins, especially short chain ones Proteohormones like endotheline, when coupling one Chelators via a covalent bond their effectiveness to lose. The behavior of the was therefore completely unexpected Compounds according to the invention in the covalent bond on endotheline, its antagonists or fragments (Example 11, 12).

The type of generation of the invention shown here according to N-alkyl complexing agents of the general formula I. Endothelines (Examples 11 and 12) from chelation Prepress is a new way to generate such complex formation sites. The stability of the new N-alkyl complexes of the general formula I correspond to the known N₃S complexes.  

Non-invasive techniques to diagnose atherosclerosis have been described, especially with a radio iodine Mark. So were labeled anti with radioisotopes body or labeled "low density lipoproteins" (LDL) presented on atherosclerotic wall areas bind (Lees et al, 1983, J. Nucl. Med. 24, 154-156, Kaliman et al, 1985, Circulation, 72, 300; Virgolini et al, 1991, Eur. J. Nucl. Med., 18, 944-947). This metho However, the include major disadvantages, such as. B. the antigenic effect of the antibodies on the organism and the long period of time (several days) required for isolation purification, labeling and labeling of the LDL from the blood of the Patient is needed. But above all, these are big ones Molecules are characterized by a long half-life in the blood draws together with a high background beam localization of the atheros throughout the body clerotic lesions difficult, if not impossible (Shih et al, 1990 Proc. Natl. Acad. Sci., 87, 1436-1440), synthesized partial sequences of the LDL protein partly (apo-B-100), although still atherosclerotic bind plaques, but by a much shorter re half-life in the blood and an improved Si Highlight signal / noise ratio. Because of one too low affinity for plaque and / or low Density of the binding sites of these peptides in the plaque, could not be successful with these apo-B peptides either in vivo diagnosis of atherosclerosis can be shown.

In general, these compounds have the disadvantage of radioactive iodine-123. The poor availability due to the production in the cyclotron and the physical half-life of 13 hours, combined with the easy release in the organism. It was therefore surprising that the compounds of the formula I according to the invention showed good accumulation in atherosclerotic plaques after covalent binding to NH groups of the endothelin and complexation with ^99m Tc. Particularly noteworthy is the advantage that the n = 1 and n = 0 compounds generate new N-alkyl-peptide complexing agents via an amide formation after covalent binding to endotheline, possibly including the NH- and SH- Groups of the respective endothelin.

When the compounds according to the invention are reacted with dopaminergic bioactive molecules, the receptor affinity of these compounds from the ergoline series is surprisingly not lost, but it was even possible in the case of precursors - with or without affinity for the D₂ receptor - to achieve adequate brain mobility, which is a representation of the D₂ Receptors. Efforts to display receptors in the brain with ^99m Tc ⁺⁵ complexes have so far been unsuccessful. Thus, when 8α-amino-6-methyl-ergoline is reacted with mercapto-acetyl-glycyl-glycyl-glycine to form 8α-amide and complexed with ^99m Tc ⁺⁵ after iv injection in the rat (Wistar), there is no uptake in the brain .

If, on the other hand, the reaction is carried out with the ß compounds according to formula I, wherein R¹ = methyl and the amidation or esterification takes place over R², so you get a rich under analog conditions tion in the brain from 0.1 to 0.5% of the dose.

With n = 1 and n = 0 complexes, new N-alkyl Peptide complex structures that work together with the bioactive molecule, surprisingly structure mimicking ren dopaminergic compounds and a high Enrichment in the brain show the visualization the D receptors can be used (Formula IX).

The preparation of the compounds according to the invention follows according to the methods known to the person skilled in the art [A Spe cialist Periodical Report; Amino acids, peptides and Protein; The Chemical Society, Burlington House, London, W1VOBN]. To prepare the compounds with R¹ = CH₃ and R² = OH you start from the Sarcosin and put it with Chloroacetyl chloride. The resulting chloroacetyl sarco sin is reacted with thiobenzoic acid in the usual way and you get structures with n = 0

Compound A

The compounds with n = 2 are produced by Implementation of the diketopiperazine of glycine with Chloroacetyl chloride. The resulting chloroacetylglycyl  Glycine is then used with N-methylamine or longer residues with N-alkylamines, phenylamines, Alkyl-oxa-alkylamines, cycloalkylamines or Glycinglycolic acid ester implemented. By repositioning with chloroacetyl chloride and subsequent thiobenzoyly tion of the formula n = 2 are obtained.

Compound B

R¹ stands for the variations on the sarcosyl stick substance atom.

The corresponding compounds with n = 1 are obtained by reacting salts of glycine with N-protected sarcosyl compounds or by reacting glycine with chloroacetyl chloride with subsequent aminolysis of the Chloromethyl group. Reaction with chloroacetylchlo rid and subsequent thiobenzylation provides the Title compounds with n = 1.

Compound C

The compounds A, B, C react in the usual way Amino group or hydroxyl group-carrying reaction partners. For this purpose, A to C are in N-methyl pyrrolidone dissolved, with BOP (B. Castro et al Tetrahedron Letters 14 (1975) 1219) or TBTU (Knorr et al Tetrahe dron Letters 30 (1989), 1927) preactivated and then implemented with the amino component. Esterifications  are preferably counteracted with dicyclohexylcarbodiimide were carried out by dimethylaminopyridine. For the Synthesis of compounds with an estrogen as Bioli gand one assumes an estrogen that already has one unsaturated substituents in 17α such as propargylamine or Propargylyalkoholrest carries and sets this with one of the compounds A to C in the manner described around.

The reaction of compounds A to C with peptides, in particular with endothelin building blocks takes place in the Peptide chemistry conventional methods either conventional nell in solution, preferably in dimethylformamide, dime thylacetamide, N-methylpyrrolidone, dimethyl sulfoxide or Mixtures of these components. When using the Solid phase method can acylation of the amino component nente also under conventional conditions on the synthetic resin respectively. The ge wanted connections. A preparative cleaning, if required, carried out chromatographically on an RP 18 Column in a water / acetonitrile gradient that is 0.1% Contains trifluoroacetic acid.

For reaction with amino groups or hydroxyl groups carrying ergolines was analogous to the implementations Move peptides in solution. The implementations were in N-methylpyrrolidone performed. The preparative Reini is also carried out as indicated for peptides.

The other R⁴ residues are protected after building up Obtain dipeptides A-C in which R¹, where R² = OH, after activation with

NH₂-CH₂Cl,

NH₂-CH₂-CH = CHJ or

NH₂-CH₂-C = CJ

is implemented and after the palladium-O reaction the 17α- Ethinyl steroid with the desired complexing agent a C-C bond is covalently bound.

The complex formation is preferred in an aqueous medium, at a pH between 6 and 9 and at room temperature tur, by reduction of Na pertechnetate with z. B. Tin (II) chloride or dithionide, if necessary via an auxiliary such as sodium citrate or sodium tartrate, the protective group R³ previously or in situ is split off.

An activation of the R² that arises in the case of R² = OH Carboxylgruppe or the replacement of the carboxyl group by an NCS or similar reactive group cause Peptide chelates covalently to a larger protein or Proteohormones, Endotheline, Endothelin Analogs, Endothe lin antagonists, endothelin derivatives, partial sequences of Bind endothelins and subsequently a complexy implementation.

As indicated in the preparation instructions, the ^99m Tc and ^186.188 Re complexes ^according to the invention can be obtained in 95% yields at room temperature, neutral to weakly alkaline pH and with or without re-chelation (e.g. via a heptagluconate- ^99m Tc complex) produce as complexes. Their slight salt formation via a free cation leads to good water solubility.

Production of the metal complexes: the splitting off of the Protecting group takes place according to methods known to the person skilled in the art (see "Protective groups in organic synthesis" T. N. Greene, John; Wiley and Sons 1981).  

For use in human diagnostics usually used between 370 MBq and 1850 MBq, preferably 740 to 1480 MBq. For human use the compounds of the invention are in the form of a Kit’s provided. The kit contains at least one Chelator according to the general formula I in free or on Ligand bound form.

Another object of the invention is a cold kit, which a chelating agent according to the general formula I in free form or to a bioactive molecule, such as. B. an ergoline, estradiol or endothelin derivative which contains and which is capable of metal atoms tie.

The spin-off is often very difficult the intermediate introduced to carry out the synthesis Protecting groups. Especially chelating agents with peptide linkage are removed when the -S protective group (R³) is split off by simultaneous cleavage of the peptide chain badly affected or do not lead to more usable fragments, the more surprising it that with the simultaneous presence of water substance / Pd / CaCO₃, thus a deprotection under hydrating conditions, in the presence of the usual applied alkali, a high one, reaching up to 80% Spin-off occurs.

This crucial improvement in the synthesis of the free chelate complexes is of great importance as a deprotection in front of the kit form lation can take place and the protection group pen-free chelators is technically significantly improved.  

Using example 3, the cleavage method according to the invention de described. While with the usual spin-off Alkali obtained a mixture that is difficult to separate was obtained when the protecting groups were split off Alkali in the presence of hydrogen / Pd / CaCO₃ a 100% Yield with over 80% of the desired material.

An example is that the reaction with amines or alcohols via an activation of the carboxylic acid R² = OH runs, methods known to those skilled in the art be applied.

Examples of activated groups are: Säu rechloride, mixed anhydrides [Org. Prep. Proc. Int. 1975, 7, 215], activated esters [Adv. Org. Chem. Part B, 472]. Linking with biomolecules can be direct or also done via linker, here is the carbodiimide metho de (Fieser, Reagents for Organic Synthesis 10, 142) called. The linkage is such that between Chelate and biomolecule formed a covalent bond becomes.

When using steroids as biomolecules prefers estradiol derivatives with substituents in 17α- Position used. The synthesis of such compounds z. B. von Blickenstaff for 17α-ethynyl derivatives of Estradiols described. (Blickenstaff A .; Steroids 46, 889 (1985); USP 462 426). For estradiol derivatives with 17α- Propargyl substituents with terminal NH₂ or OH function is the synthesis of Blickenstaff, Brandes and Poirier. (Blickenstaff et al; Steroids 48, 223 (86); Brandes, A. et al Dissertation 87-01 441, 1986, University of Illinois, D. Poirier et al, J. Steroids. Biochem. Molec. Biol. 38, No. 6, 759-774, 1991).  

An overview of the combination of triple with Double bonds, e.g. B. after the palladium-O reaction, are in Aldrichchimica Acta, Vol. 15, No. 1, 1982; Shun ichi Murahashi et al, Stephen A. Godleski, Tetrahedron Letters, Vol. 22, No. 24, pp 2247-2250, 1981; Tetrahedron Letters, Vol. 29, No. 24, 2973-2976, 1988; described. Here, for. B. I with R² = -NH-CH₂-C≡CH in the Kopp lung with z. B. 17α [2-halogeno-ethenyl] -estradiol according to mentioned references implemented.

It is known from the literature that ergot alkaloids of the type the 6-methyl-8-substituted ergoline over a wide range Spectrum of chemical variation, without that today a connection between effect and substituents would be clearly recognizable in position 8 [Ergot Alkaloids and Related Compounds, Editors: B. Berde and H. O. Schild, Springer-Verlag, Berlin, Heidelberg, New York, 1978].

A preferred dopaminergic ligand is ergoline. A Overview of synthetic routes to 8-substituted ergolines can be found at Ergot Alkaloids and Related Compounds, Editors B. Berde and H. O. Schild, Springer-Verlag Berlin, Heidelberg, New York, 1978, Chapter II Chemical Back ground, J. Ruisemmann and P.A. Stadler.

The reaction with the 8α-amino or 8β- Hydroxy-methyl-ergoline. It takes place via an activated Group in R², for example that with benzotriazol-1-yl tetra-methyl-uronium tetrafluoroborate or after known carbodiimide method.

When using n = 1 and n = 0 in the application according to the invention Complex or partial complexing agents are included drawing of nitrogen in the 8α-amino and 6-N-methyl Ergoline group generated new complexes.  

Another object of the invention is the use the metal chelate for diagnostic and / or therapeutic cal purposes as well as pharmaceutical means that at least a chelate according to the invention of the general formula I, if necessary with the additives common in galenics, contain.

For use in humans, the invention Connections in the form of a cold or hot kit posed. The kit contains at least one chelator after Formula I in free or bound form, the Ligan preferred from the class of Ergoline, Estra diols, endotheline, short-chain synthetic peptides, Cholecystokinins can be obtained.

Examples General complex formation with sodium gluconate ^99m

Tc gluconate: 2 ml ^99m

Tc generator eluate are added to 2 ml of 0.1 M sodium gluconate solution and mixed with 10 ul 0.01 M SnCl₂ (in 0.01 M HCl). The quantitative pertechnetate reduction is then checked by thin-layer chromatography.
TLC (silica gel / acetone): ^99m

Tc gluconate Rf: 0-0.1; ^99m

TcO⁴-Rf: 0.9

example 1 [ ^99m Tc] mercaptoacetyl sarcosine 1st stage: chloroacetyl sarcosine

0.07 mol sarcosine are dissolved in 50 ml 1N sodium hydroxide solution and at 0 ° C in portions with 0.08 mol chloroacetyl chloride  and 110 ml of 1N sodium hydroxide solution were added. After 45 min The addition is ended and the reaction solution is mixed with 20 ml of 5N Acidified hydrochloric acid.

The product is evaporated to dryness under reduced pressure and the residue is extracted three times with 50 ml of cold acetone each time. The acetone is removed under reduced pressure and the residue is extracted with 100 ml of ether. The ether is evaporated off and the oil which remains is taken up in a little ether and brought to crystallization with vigorous stirring.
Yield: 77% of theory Th.
TLC: silica gel // butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.65 (iodine vaporization).

2nd stage: benzoylmercaptoacetyl sarcosine

0.02 mol of chloroacetyl sarcosine in 750 ml of methanol with stirring in a protective gas atmosphere at room temperature rature solved. A solution of 0.041 mol is slowly added Thiobenzoic acid, dissolved in 20 ml of methanol and neutral Siert with sodium methylate, added dropwise and another 12 h touched.

The solvent is removed under reduced pressure and the residue was taken up with 2N hydrochloric acid. The The reaction product precipitates as a brown oil, which is derived from the the supernatant solution separated and with extra chloroform is hated. The chloroform is made under reduced pressure removed and the residue mixed with ether. By The reaction product becomes cool and vigorous stirring precipitated, filtered off and dried on the clay plate.  

pH titration and IR spectrum showed that the reaction product from a mixture of 60% methyl ester and 40% free amino acid existed.

3rd stage: splitting off of the protective group

0.08 mmol of the mixture obtained are absolute in 2 ml tem suspended methanol, with 2 mg Pd / CaCO₃ (5%) ver sets and under a hydrogen pressure of 66 kPa Stir with a solution of 0.13 mmol sodium methylate in 1 ml of absolute methanol implemented. The mixture is stirred for a further 15 min and then neutralizes the solution with methanolic Dowex 50WX8. The catalyst and the resin are filtered off tried, the substance lyophilized and the contaminants extracted with 2 ml of benzene. Then that will Benzene separated and the substance lyophilized from ethanol siert.

The resulting product mixture of ester and free acid is separated on Sephadex G10. (14 × 1000 column, 20 ml / h, RI detector) Mercapto-acetyl-sarcosine methyl ester is separated as the second fraction, cf. Example 1 a).
Mercaptoacetyl sarcosine:
TLC: silica gel // butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.7 (ninhydrin)
RP 18 / methanol Rf: 0.85 (ninhydrin)
1 H-NMR (DMSO) TMS δ2.9 (SH, 1H), δ3.1 (N-CH3, 3H), δ3.9 (-CH2, 2H)

Labeling of N-methyl-MAG1

About 1 mg of N-methyl-MAG1, dissolved in 200 µl of water, is added to 2 ml of ^99m Tc-gluconate solution. After a reaction time of 15-20 min, the purity is checked by TLC on silica gel 60/95% ethanol.

The main amount of activity runs with Rf = 0.1 (approx. 80%); further peaks at Rf = 0.2 (approx. 10%) and Rf = 0.4 (approx. 10%). An anionic Tc-MAG1 complex is detected in the electropherogram with a relative mobility ^u pertechn./ ^u TcMAG1 of 0.2.

Example 1a

When separating the reaction mixture from example 1, level 3 you get 20% mercapto-acetyl-sarcosine methyl ester as a colorless oil.

Example 2 Mercapto-acetyl-hexylaminoacetic acid 1st stage: hexylaminoacetic acid

0.021 mol of chloroacetic acid are mixed with 0.1 mol of hexylamine and left to stand for 5 days at room temperature. The thick syrup is then stirred into 500 ml of acetone and crystallized in the form of white leaves.
Yield 2.1 g (63% of theory)
TLC: silica gel // butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.6 (ninhydrin).

2nd stage: chloroacetyl-hexylaminoacetic acid

0.01 mol of hexylaminoacetic acid are dissolved in 10 ml of 1N sodium hydroxide lye dissolved, cooled and alternately at 0 ° C with 1.5 ml (1 ml = 0.012 mol) chloroacetyl chloride and 20 ml 1N Na  tron lye added. The reaction is complete after 30 min and heating is continued for 30 minutes with gentle heating stirs. It is acidified with 3 ml of 5N hydrochloric acid and that precipitating brown oil separated.

The oil is extracted several times with hot water and used as a colorless oil for the next synthesis step without further purification.
TLC: silica gel // butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.75 (iodine vaporization).

3rd stage: Benzoylmercaptoacetyl-hexylaminoacetic acid

0.018 mol of chloroacetyl-hexylamino acid are stirred in 500 ml of methanol under nitrogen. 0.036 mol of thiobenzoic acid are neutralized with sodium methylate and added to the solution within 30 minutes. The mixture is stirred under protective gas for a further 12 h. The methanol is removed under reduced pressure, the residue is acidified with 2N hydrochloric acid and the yellow oil which separates out is separated.
Yield: 370 mg
TLC: silica gel // butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.5

Example 3 [ ^99m Tc] mercaptoacetyl sarcosyl diglycine 1st stage: chloroacetyl diglycine

10 g of glycine anhydride are dissolved in a 250 ml three-necked flask in 50 ml of 2N sodium hydroxide solution at room temperature. After 45 minutes, the solution is cooled to 0 ° C. and 11.1 g of chloroacetyl chloride and 24 ml of 5N sodium hydroxide solution are added dropwise with continued stirring and cooling. The addition is complete after 45 min. Then 40 ml of 5N hydrochloric acid are added, the solution decolorizing and the substance starting to crystallize. It is left to stand at 0 ° C. for about 1 h and suction filtered. It is washed several times with cold water and recrystallized from hot water.
Yield: 5 g.

The mother liquor is concentrated under reduced pressure. The precipitate is recrystallized from water.
Yield: 2.5 g
Mp: 173 ° C
TLC: silica gel // n-butanol / glacial acetic acid / water / 4: 1: 1
Rf: 0.75 (iodine vaporization)
IR (fixed in KBr): ν _{C = O} 1636, 1676; δ _NH 1550; ν _COOH 1708 cm ^-1 .

2nd stage: sarkosyl diglycine

5 g of chloroacetyl diglycine are mixed with 15 ml of 33% aqueous methylamine solution and left for 2 days at room temperature. The solution is then concentrated to thick syrup under reduced pressure, the syrup is heated on a water bath and mixed with 50 ml of hot ethanol. The precipitate is allowed to settle in the cold and is suctioned off through a G4 frit. It is dissolved in 43 ml of hot water and 43 ml of hot ethanol are added. The product crystallizes out, the product is filtered off with suction and lyophilized.
Yield: 3 g = 60% of theory. Th.
Mp: 237 ° C
TLC: Silufol // n-butanol / glacial acetic acid / water / 4: 1: 1
Rf: 0.3 (ninhydrin)
IR (fixed in KBr): ν _{C = O} 1620, 1650, δ _NH 1540; ν _COOH 1672 cm ^-1
1 H-NMR (DMSO) TMS δ 2.8 (N-CH₃, 3H), δ 3.8-4.1 (-CH₂-, 6H).

3rd stage: chloroacetyl-sarkosyl-diglycine

2 g of sarkosyl diglycine are dissolved in 10 ml of 1N NaOH at room temperature and then alternately mixed with 0 ml of chloroacetyl chloride and 16 ml of 1N NaOH at 0 ° C. within 30 minutes. The mixture is stirred for a further 30 min, acidified with 3 g of 5N HCl and the product is evaporated to dryness under reduced pressure. The resulting crystals are dissolved in hot water and ethanol is added to the solution. The sodium chloride which has precipitated is filtered off and the filtrate is concentrated under reduced pressure. The desired product is extracted by repeated treatment with 30 ml of hot acetone each time, the acetone is removed under reduced pressure and the residue is taken up in a little water and lyo philized.
Yield: 1.1 g = 53% of theory. Th.
Mp: 74 ° C
TLC: Silufol // n-butanol / glacial acetic acid / water 4: 1: 1
Rf: 0.7 (iodine vaporization)
IR (fixed in KBr): ν _{C = O} 1655; δ _NH 1550; ν _COOH 1730 cm ^-1 .

4th stage: Benzoylmercaptoacetyl-sarkosyl-diglycin

1 g of chloroacetyl-sarkosyl-diglycin is in 140 ml of Met hanol, p.a., with stirring and under protective gas at room temperature solved.

In an Erlenmeyer flask, 0.8 g of thiobenzoic acid are dissolved in 3 ml of methanol and neutralized with sodium methylate. This solution is slowly added dropwise to the starting solution with stirring and under a protective gas atmosphere, and the mixture is stirred for a further 12 hours. The solvent is removed under reduced pressure and the residue is taken up in 2N HCl. It is suctioned off and washed neutral with warm water. The mixture is then washed with 20 ml of chloroform, 20 ml of acetonitrile and 20 ml of ether and the residue is dried under reduced pressure.
Yield: 0.83 g = 61% of theory
Mp: 134 ° C
TLC: Silufol // n-butanol / glacial acetic acid / water 4: 1: 1
Rf: 0.8 (iodine vaporization)
IR (fixed in KBr): ν _{C = O} 1620; δ _NH 1550 cm ^-1 , ν _COOH 1720 cm ^-1 .

5th stage: mercaptoacetyl-sarkosyl-diglycin Alkaline removal of the protective group

The protective group is split off according to the usual procedure Ren with sodium methylate led to a very unsettled final product. The mercaptoacetyl-sarkosyl-diglycin is only included to about 25%. In addition to a not identi Defected by-product arises during this saponification the main thing is mercaptoacetyl sarcosine and diglycine. The desired product is obtained only in low yield ten.

Alkaline elimination under hydrogenation conditions

0.08 mmol substance are in 2 ml of anhydrous methanol suspended, mixed with 2 mg Pd / CaCo₃ (5%) and at a hydrogen pressure of 60 kPa with stirring Solution of 0.13 mmol sodium methylate in 1 ml of anhydrous Methanol implemented. The mixture is stirred for 15 minutes and neutral The solution is then dosed with methanolic Dowex 50WX8. The catalyst and resin are filtered off Substance lyophilized and extracted with 2 ml of benzene.  

The benzene is then separated off and the substance is lyophilized from ethanol.
Yield: 10.2 mg = 61% of theory. Th., Colorless oil
TLC: Silufol // n-butanol / glacial acetic acid / water 4: 1: 1
Rf: 0.4 (ninhydrin)
RP 18 // methanol
Rf = 0.8 (ninhydrin)
IR (fixed in KBr): ν _{C = O} 1650, 1680; δ _NH 1550, ν _COOH 1745 cm ^-1
1 H-NMR (DMSO) TMS δ 2.8 (SH, 1H), δ 3.0 (N-CH₃, 3H), δ 3.5- 4.1 (-CH₂, 8H), δ 8.1-8 , 2 (CO-NH-, 2H)

[ ^99m Tc] mercaptoacetyl sarcosyl diglycine

1 ml of ^99m Tc-gluconate solution is mixed with a solution of 0.4 mg of pure substance from precursor 4 in 0.5 ml of water. The reaction is complete after 30 minutes.
TLC: (silica gel 60, 95% ethanol):
Two components Rf 0-0.1 (40%), Rf 0.8 (60%)
Electrophoresis (pH 7.0):
Both components migrate as anions.

Example 4 Synthesis of [ ^99m Tc] -Mercaptoacetyl-Hexylglycyl-Diglycin 1st stage: hexylglycyl diglycine

0.0048 mol of chloroacetyl diglycine are mixed with 0.05 mol of hexylamine and 5 ml of ethanol and left to stand. After 6 days the solvent is removed under reduced pressure. The oily residue is poured into 50 ml of acetone and heated. After cooling, the yellow solution is separated off and the white residue is washed again with about 5 ml of acetone. The product is dried on the clay plate.
Yield: 610.5 mg (47% of theory)
TLC: Silufol // n-butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.3 (ninhydrin)
RP 18 // methanol, Rf: 0.7 (ninhydrin).

2nd stage: chloroacetyl-hexylglycyl-diglycine

0.0022 mol of hexylglycyl diglycine in 5 ml of 1N sodium hydroxide lye dissolved and stirred for 30 min at room temperature. The solution is then cooled. Be at 0 ° C alternately 0.3 ml of chloroacetylchlo within 30 min rid and 4 ml of 1N sodium hydroxide solution. Then turns 30 min stirred without cooling.

After acidification with 5N hydrochloric acid, the solvent is removed under reduced pressure and the product is obtained as a yellow oil. After repeated extraction with 100 ml of hot acetone each time, the acetone is removed under reduced pressure and the residue is slurried with 30 ml of acetone. The white sediment is dried on the clay plate.
Yield: 311 mg (40% of theory)
TLC: Silufol // n-butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.75 (iodine vaporization).

3rd stage: Benzoylmercaptoacetyl-hexylglycyl-diglycin

0.00089 mol of chloroacetyl-hexylglycyl diglycine are dissolved in 70 ml of methanol (especially pure for microelectronics) and stirred under protective gas (N₂). 0.002 mol of thiobenzoic acid are dissolved in 5 ml of methanol and neutralized with 0.4 ml of Na methano lat. This solution is slowly added under protective gas and stirring is continued for a further 12 hours. The methanol is removed under reduced pressure and the residue is taken up in 2N HCl. The hydrochloric acid is also removed under reduced pressure, the residue was washed with water and the wash water was removed by decanting. A little methanol is added to the viscous residue and the product precipitates. It is washed with acetronitrile and dried on the clay plate.
Yield: 253.3 mg (67% of theory)
TLC: Silufol // Butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.8 (UV).

4th stage: mercaptoacetyl-hexylglycyl-diglycine

0.064 (28.8 mg) mmol benzoylmercaptoacetyl-hexylglycyl- diglycine are suspended in 2 ml of methanol, 2.7 mg 5% Pd / CaCO3 added and attached to the hydrogenation apparatus brings. 0.04 ml of Na methano are in an angle flask lat and 1 ml of methanol. The apparatus becomes evacuated and then flushed with hydrogen.

The methanolate is added and the mixture is stirred at a hydrogen pressure of 60 kPa for 15 min. The solution is acidified with Dowex 50WX8 methanol, the resin is filtered through a pleated filter (argon bell), washed with methanol and then lyophilized. The residue is extracted three times with 4 ml of benzene, the benzene is removed by decanting, the residue is again taken up in methanol and lyophilized. The product was purified on a Sephadex column.
Yield: 9.8 mg (42% of theory)
TLC: Silufol // Butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.5 (ninhydrin)
1 H-NMR (DMSO) TMS δ 1.2 (-CH3, 3H), δ 3.1 (SH, 1H), δ 3.2 (N-CH2, 1OH), δ 3.7 (-CH2, 6H) , δ 3.8 (CH2-SH, 2H) δ 8.2-8.5 (-NH, 2H).

[ ^99m Tc] mercaptoacetyl hexyl glycyl diglycine

1 ml of ^99m Tc-gluconate solution is mixed with 400 µl 0.1 N NaOH and 30 µl precursor 4 solution - 1.63 mg / 100 µl water. After standing for 40 minutes, the reaction mixture is neutralized with 2 ml of a 0.1 M sodium phosphate buffer solution (pH 7.0). The reaction mixture is stable for at least 2 hours.
TLC (silica gel 60; 95% ethanol)
Rf: 0.7 (95%)
Electrophoresis (pH 7.0): migration as an anion

Example 4a Enrichment of [99mTc] -Mercaptoacetyl-Hexyl-glycine- Diglycine in atherosclerotic plaques from aorta WHHL rabbit

99.9 GBq (2.7 mCi) of the substance labeled according to Example 4 was diluted to 1 ml with phosphate-buffered saline and applied to an anesthetized WHHL rabbit Rom pun / Ketavet (1: 2) via an ear vein. The rabbit was sacrificed 5 hours after application and both autoradiography of the aorta and Sudan III staining were carried out to show the atherosclerotic plaques ( FIG. 1). The enrichment factor between normal and atherosclerotic wall areas was between 3 and 5 depending on the formation of the plaques (Sudan III staining). The in vivo representation of a WHHL rabbit is shown in Fig. 2.

Example 5 S-Bzl-acetyl-Sar-Gly-Gly- [8α-ergolinylamide]

200 mg S-Bzl-acetyl-Sar-Gly-Gly-OH and 161 mg Benzotria zol-1-yl tetramethyl uronium tetrafluoroborate was used in 1 ml of N-methylpyrrolidone dissolved and 172 ul diisopropyl ethylamine added. After 3 min the colorless solution becomes a stirred solution of 120 mg of 8α-amino-ergoline dripped. The red-brown solution is at room temperature stirred for a further 4 hours, the progress of the reaction HPLC chromatography on a VYDAC C18 column (4.6 × 250 mm) applying a gradient from 10% to 60% B was followed in 25 min (solvent A 1000 ml water / 2 ml trifluoroacetic anhydride, mobile phase B 500 ml Acetonitrile / 100 ml water / 1 ml trifluoroacetic acid anh drid).

Without further pretreatment, the reaction mixture is preparatively separated on a VYDAC column (40 × 300 mm) by applying a gradient from 20% to 30% B in 20 min (eluent composition as indicated above). It is then lyophilized.
Yield: 75 mg
A mass spectrum showed the expected molecular weight.

Example 6 S-Bzl-acetyl- [N1-hexyl] -Gly-Gy-Gly- [8α-ergolinylamide]

60 mg of S-Bzl-acetyl- [N1-hexyl] -Gly-Gy-Gly-OH and 40 mg of benzotriazol-1-yl-tetramethyl-uronium tetrafluoroborate were suspended in 0.6 ml of N-methylpyrrolidone and by adding 17 , 2 ul geisopropylethylamine brought in solution. This solution was added dropwise to a solution of 30 mg of 8α-amino-ergoline in 0.4 ml of N-methylpyrrolidone. After 4 hours the reaction solution was worked up. The crude reaction mixture was chromatographed preparatively (as described for Example 5). Subsequent lyophilization gave the desired product.
Yield: 30 mg of a pseudocrystalline product.
The mass spectrum showed the expected molecular weight.

Example 7 S-Bzl-acetyl-Sar-Gly- [8α-ergolinylamide]

180 mg of S-Bzl-acetyl-Sar-Gly-OH and 161 mg of benzotriazol-1-yl-tetramethyl-uronium tetrafluoroborate were dissolved in 1 ml of N-methyl-pyrrolidone and 172 μl of diisopropylethylamine were added. After 3 minutes, the colorless solution is added dropwise to a solution of 120 mg of 8α-amino-ergoline with stirring. The red-brown solution is stirred at room temperature for 4 hours and - as described in Example 5 - worked up and chromatographed.
Yield: 70 mg (pseudocrystalline)
An MS showed the expected peak.

Example 8 S-Bzl-acetyl-Sar- [8α-ergolinyl-amide]

120 mg S-Bzl-acetyl-Sar-OH and 161 mg benzotriazol-1-yl- tetramethyl uronium tetrafluoroborate were dissolved in 1 ml of N methyl-pyrrolidone dissolved and 172 ul diisopropylethylamine added.

The reaction is then carried out analogously to example 5 and Processing and separation of 52 mg of a pseudocrystalline Connection.

An MS shows the expected mass peak.

Example 9 3,17β-dihydroxy-17α- [5 (S-benzoylthio-acetyl-sarkosyl-glycyl-) amino-pent-1-en- 3-in] -1,3,5-estratriene

It is added to a suspension of 130.0 mg of 17β-hydroxy-17α-iodovinyl-1,3,5-estratriene-3-tetrahydropyranyl ether, 0.35 g [S-benzoyl-thioacetyl-sarkosyl-glycyl propargylamide], 11. 0 mg of benzyl-triethylammonium chloride, 11.5 mg of tetrakis (triphenylphosphine) palladium (0), 9 mg of copper (I) iodide, suspended in 5 ml of toluene and stirred for 90 hours at room temperature. Water is added, the mixture is extracted with toluene and the mixture is dried. The solvent is removed under reduced pressure, the residue is taken up in 10 ml of tetrahydrofuran, 190 mg of pyridinium paratoluenesulfonic acid in 5 ml of ethanol are added and the mixture is heated under reflux for 3 hours. The solvent is then removed under reduced pressure and the mixture is purified on a silica gel column using methylene chloride / methanol (8: 1).
Yield: 30% of theory Th.
The substance shows an Rf of 0.4 in TLC (CH₂Cl₂ / MeOH] 5: 5.

Example 10 3,17β-dihydroxy-17α- [N- (benzoyl-thio-acetyl-sarkosyl-glycyl) -amino-propyne-1] -1,3,5-estr-atriene

It is added to a suspension of 110 mg of 17β-hydroxy-17α- propargyl-amino-1,3,5-estratriene-3-tetrahydropyranyl ether, in 3 ml DMF 350 mg S-benzoyl-thioacetyl-sarkosyl- glycine in 3 ml DMF. The mixture is stirred at 100 ° C. for 24 hours Shielding gas.  

After adding 10 ml of tetrahydrofuran, one sucks Crystallized and concentrated under reduced pressure. The residue is taken up in 20 ml of tetrahydrofuran, with 100 mg pyridinium-para-toluenesulfonic acid in 3 ml Dissolved ethanol, added and refluxed for 1 hour heated. After removing the solvent on silica Low pressure gel column in the methanol / methylene chloride system (1: 8) cleaned.

The substance showed an Rf of 0.6 in TLC (CH₂Cl₂ / MeOH) 5: 5.
Yield: 71 mg

Example 11

0.001 mol S-Bzl-acetyl-Sar-Gly-OH and 322 mg Benzotria zol-1-yl-tetramethyl uronium tetrafluoroborate are in 3 ml of DMF dissolved with the addition of 344 µl of diisopropylethyl min. It is used to form the benzotriazolyl ester 5 Preactivated minutes and then the clear solution 33 mmol protected His (Trt) -Leu-Asp (OBut) -Ile-Ile-Trp- Resin added. The suspension is stirred for one hour then washed several times with DMF, suction filtered and dried. The dried resin is then used as usual TFA / Scavanger treats the product from protection free groups. The cleaning becomes like for example 5 described.

The compound obtained according to Example 10 is complexed with ^99m Tc by the method given as the general method (Example 1).

Example 12

0.010 mol His (Trt) -Leu-Asp (OBut) -Ile-Ile-Trp-OH, herge exhibits on Sasrin resin, are in a mixture DMF / NMP with the addition of 0.010 mol diisopropylethylamine dissolved and with stirring with 0.010 mol S-Bzl-acetyl-Sar- OH, prepared as in Example 11, dissolved in 3 ml DMF transferred. It is stirred for 2 hours and then that Solvent removed. The remaining residue will stirred with water and suction filtered, washed and dried net. The protective groups are removed as usual and that Obtain product by pouring into ether. The cleaning is carried out as described for Example 5.

S-Bzl-acetyl- [N14-bromophenyl] -Gly-Gly-Gly-OH 1st stage: chloroacetyl diglycine

10 g of glycine anhydride are dissolved in a 250 ml three-necked flask in 50 ml of 2N sodium hydroxide solution at room temperature. After 45 minutes, the still somewhat cloudy solution is cooled to about 0 ° C. and 11.1 g (7.8 ml) of chloroacetyl chloride and 24 ml of 5N sodium hydroxide solution are added dropwise with stirring. The addition is complete after 45 min. The solution turns pink. Then 40 ml of 5N hydrochloric acid are added, the solution decolorizing and the substance starting to crystallize. The mixture is left to stand at 0 ° C. for about 1 hour and the precipitate is suctioned off. It is washed several times with cold water and recrystallized from hot water.
Yield: 5 g
Mp: 173-174 ° C.

The mother liquor is concentrated under reduced pressure and the residue is also recrystallized from water.
Yield: 2.5 g
Mp: 173 ° C
Overall yield: 7.5 g = 41% of theory
TLC: Silufol // n-butanol / glacial acetic acid / water 4: 1: 1.

2nd stage: 4-bromophenyl-triglycine

0.0144 mol of chloroacetyl diglycine are refluxed with 0.029 mol of 4-bromoaniline in 20 ml of ethanol and 20 ml of 1N NaOH for 7 hours. After removing the solvent under reduced pressure, the remaining residue is extracted three times with 50 ml of warm acetone each. The product to be left behind is recrystallized from hot water. The crude product is used in stage 3.
Yield: approx. 60%.

3rd stage: chloroacetylation of 4-bromophenyl-triglycine

0.01 mol of 4-bromophenyl-triglycine is dissolved in 10 ml of 1N NaOH at room temperature and then 0.012 mol of chloroacetyl chloride and 20 ml of 1N NaOH are alternately added at 0 ° C. with stirring within 30 minutes. The mixture is stirred for a further 30 min, acidified with 5N HCl and the product is evaporated to dryness under reduced pressure. The resulting crystals are dissolved in hot water and ethanol is added to the solution. The precipitated sodium chloride is filtered off and the filtrate is evaporated on a rotary evaporator. The desired product is extracted by repeated treatment with 30 ml of hot acetone each time, the acetone is removed under reduced pressure and the residue is taken up in a little water and lyo philized.
Yield: 50% of theory
TLC: Silufol // n-butanol / glacial acetic acid / water 2: 1: 1
Rf: 0, 6.

4th stage: S-Bzl-acetyl- [N¹-bromophenyl] -Gly-Gly-Gly-OH

0.01 mol of the chloroacetyl product are dissolved in 140 ml of methanol with stirring and protective gas at room temperature. For this purpose, 0.02 mol of thiobenzoic acid is dissolved in 20 ml of methanol and neutralized with sodium methylate, slowly added dropwise with stirring and under a protective gas and stirred for a further 12 hours. The solvent is removed under reduced pressure and the residue is taken up in 2N HCl. The resulting crystals are suctioned off and washed neutral with warm water. The mixture is then washed with 20 ml of chloroform, 20 ml of acetonitrile and 20 ml of ether and the residue is dried under reduced pressure.
Yield: 60% of theory

5th stage: Thio-acetyl- [N¹-bromophenyl] -Gly-Gly-Gly-OH

0.08 mmol of substance from stage 4 are suspended with 2 ml of anhydrous methanol, 2 mg of Pd / CaCO3 (5%) are added, and at a hydrogen pressure of 66 kPa with stirring with a solution of 0.13 mmol of sodium methylate in 1 ml absolute methanol implemented. The mixture is stirred for a further 15 minutes and then the solution is neutralized with methanolic Dowex 50WX8. The catalyst and the resin are filtered off, the substance is lyophilized and extracted with 2 ml of benzene. The benzene is then separated off and the substance is lyophilized from ethanol. The cleaning is done by preparative HPLC.
TLC: Silufol // Butanol / glacial acetic acid / water 2: 1: 1
Rf: 0.8
Yield: 40% of theory

^99m Tc- [thioacetyl- [N¹-4-bromophenyl] -Gly-Gly-Gly-OH

As described for the general complexation prior to Example 1, thioacetyl- [N¹-bromophenyl] -Gly-Gly-Gly-OH is reacted with a [ ^99m Tc] gluconate preparation.

Claims (19)

1. Compounds of the general formula I R³-S-CH₂-CO-NR¹- [CH₂-CO-NH] _n -CH₂-CO-R² (I) in which
n represents a number 0, 1 or 2,
R¹ is a straight-chain or branched alkyl radical having 1 to 20 carbon atoms, which is optionally interrupted or substituted by one to three oxygen atoms, and which optionally carries a terminal -COOH, -OH or -NH₂ group, which optionally with glycolic acid or glycolic acid esters or ether is esterified or etherified,
the esters or ethers being formed with carboxylic acids or alcohols having up to 18 carbon atoms,
or represents a phenyl or cyclohexyl radical, which cher optionally in the 4-position with a COOH, NH₂ or OH group, which is optionally esterified, etherified or amidated with carboxylic acids or alcohols having up to 6 carbon atoms or substituted with a halogen atom is
R² represents a halogen atom, a halogenomethyl, methyl carboxyl, trifluoromethylcarboxyl, an NH₂ or an OH group,
the carboxyl group formed in the case of R 2 = OH either directly or after esterification with an α, ω-hydroxycarboxylic acid having up to 8 carbon atoms via its terminal carboxyl group with a biomolecule, a steroid, an ergoline derivative, a benzodiazepine derivative, a cholecystokinin, a peptide, a protein, a proteohormone, an aminosugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment is esterified or amidated,
a radical of the general formula II or the general formula IIa is where
R⁴ is a methylene, propenylene amino, propynylene amino, methylene amino or methyleneoxy group and
R⁸ represents a hydrogen atom or a methyl group,
a radical of the general formula III or the general formula IIIa represents what
R⁵ is an -NH-, -NH-CO-N <, -NH-CO-NH- or methylene oxy group,
R⁶ is a hydrogen atom, a halogen atom or a methyl group and
R⁷ represents a hydrogen atom or a straight-chain or branched alkyl radical having up to 6 carbon atoms,
R³ represents a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethylacetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or a readily removable sulfur protective group
and their salts with pharmaceutically acceptable acids or bases. 2. Compounds according to claim 1, characterized in that R¹ is a straight chain or branched alkyl group with 1 to 6 carbon atoms or a p-bromophenyl rest is. 3. Compounds according to at least one of claims 1 and 2, characterized in that R² is an OH- or CH₃-O- Represents group. 4. Compounds according to at least one of claims 1 and 2, characterized in that R² is a radical of the general formula II or the general formula IIa is where
R⁴ is a methylene or propynylene amino group and
R⁸ represents a hydrogen atom. 5. Compounds according to at least one of claims 1 and 2, characterized in that R² is a radical of the general formula III or the general formula IIIa is where
R⁵ is an NH group,
R⁶ is a hydrogen atom or a methyl group and
R⁷ represent a methyl, ethyl or n-propyl radical. 6. Compounds according to at least one of claims 1 to 5, characterized in that R³ is a hydrogen atom or is a benzoyl residue. 7. Compounds according to claim 1, namely
17α- [5- (mercapto-acetyl-sarcosyl-glycyl-amino-1-penten-3-ynyl) estra-1,3,5 (10) -triene-3,17β-diol,
6-N-methyl-8α-amino- [8α-N- (thio-acetyl-sarkosyl-glycyl-glycyl)] ergoline,
6-N-methyl-8α-amino- [8α-N- (thio-acetyl-sarkosyl)] ergoline,
6-N-methyl-8β-hydroxymethylene- [O- (thio-acetyl-sarkosyl-glycyl-glycyl)] ergoline and
6-N-methyl-8β-hydroxymethylene- [O- (thio-acetyl-sarkosyl-glycyl] ergoline. 8. Metal chelate complexes of radioactive metal ions of the elements Tc, Re, Cu, Ga, Gd, Y and In with
Compounds of the general formula I R³-S-CH₂-CO-NR¹- [CH₂-CO-NH] _n -CH₂-CO-R² (I) in which
n represents a number 0, 1 or 2,
R¹ is a straight-chain or branched alkyl radical having 1 to 20 carbon atoms, which is optionally interrupted or substituted by one to three oxygen atoms, and which optionally carries a terminal -COOH, -OH or -NH₂ group, which optionally with glycolic acid or glycolic acid esters or ether is esterified or etherified,
the esters or ethers being formed with carboxylic acids or alcohols having up to 18 carbon atoms,
or represents a phenyl or cyclohexyl radical, which cher optionally in the 4-position with a COOH, NH₂ or OH group, which is optionally esterified, etherified or amidated with carboxylic acids or alcohols having up to 6 carbon atoms or substituted with a halogen atom is
R² represents a halogen atom, a halogenomethyl, methyl carboxyl, trifluoromethylcarboxyl group, an NH₂ or an OH group,
where the carboxyl group formed in the case of R 2 = OH either directly or after esterification with an α, ω-hydroxycarboxylic acid having up to 8 carbon atoms via its terminal carboxyl group with a biomolecule, a steroid, an ergoline derivative, a benzodiazine derivative, a cholecystokinin, a peptide, a protein, a proteohormone, an aminosugar, an endothelin, an endothelin derivative, an endothelin antagonist or an endothelin fragment is esterified or amidated,
a radical of the general formula II or the general formula IIa is where
R⁴ is a methylene, propenylene amino, propynylene amino, methylene amino or methyleneoxy group and
R⁸ represents a hydrogen atom or a methyl group,
a radical of the general formula III or the general formula IIIa represents what
R⁵ is an -NH-, -NH-CO-N <, -NH-CO-NH- or methylene oxy group,
R⁶ is a hydrogen atom, a halogen atom or a methyl group and
R⁷ represents a hydrogen atom or a straight-chain or branched alkyl radical having up to 6 carbon atoms,
R³ represents a hydrogen atom, an acetyl, benzoyl, p-methoxybenzyl, acetamidomethyl, benzamidomethyl, trimethylacetamidomethyl, hydroxyacetyl, ethoxyethyl, ethylthio, trityl or an easily split off sulfur protecting group
and their salts with pharmaceutically acceptable acids or bases. 9. metal chelate complexes according to claim 8, characterized records that the radioactive metal ions are isotopes of the elements Tc and Re.   10. Metal chelate complexes according to at least one of the types Proverbs 8 and 9, characterized in that the Technetium-99m radioisotope. 11. Compounds according to claim 8, namely
[ ^99m Tc] mercaptoacetyl sarcosine,
[ ^99m Tc] mercaptoacetyl sarcosyl diglycine,
[ ^99m Tc] -Mercaptoacetyl-hexyglycyl-diglycine and
[ ^99m Tc] -Mercaptoacetyl-N¹- [4-bromophenyl] glycyl-glycyl-glycine. 12. Conjugates containing compounds of the general Formula I or metal chelate complexes radioactive Metal ions of the elements Tc, Re, Cu, Ga, Gd, Y and In with compounds of general formula I and themselves Selectively enriching substance in diseased tissues zen, with a covalent bond between them exists and this in the case of carboxy or amino group-containing substances such as peptides, Pro stones, antibodies or their fragments, amidic or in the case of hydroxyl-containing sub punch like fatty alcohols, ester-like or in the case imidic of substances containing aldehyde groups is present. 13. Conjugates according to claim 12, characterized in that that the sub. accumulating in the diseased tissue punch peptides such as endotheline, partial sequences of Endothelins, endothelin analogs, endothelin derivatives or endothelin antagonists. 14. Conjugates according to claim 12, characterized in that the peptides have the following sequences exhibit. 15. A process for the preparation of a compound of general formula I, characterized in that

• a) a diketopiperazine derivative of glycine or
• b) glycine

with a halocarboxylic acid halide and then with an amine of the general formula VI R¹-NH₂ (VI) where R¹ has the meaning given in claim 1, reacted again with a halocarboxylic acid halide and then with a compound of the general formula IVR³-SH (IV) wherein R³ has the meaning given in claim 1,
implements
or a compound of the general formula VNH₂-CH₂-CO-R² (V) wherein R² has the meaning given in claim 1, with a halocarboxylic acid halide and then with an amine of the general formula VIR¹-NH₂ (VI) wherein R¹ has the claim 1 has the meaning indicated, reacted again with a halocarboxylic acid halide and then with a compound of the general formula IVR³-SH (IV) where R³ has the meaning given in claim 1, and that this compound is optionally reacted with a pharmaceutically acceptable acid or base in whose salt is transferred. 16. A process for the preparation of metal chelate complexes of radioactive metal ions of the elements Tc and Re with compounds of the 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 I R³-S-CH₂-CO-NR¹- [CH₂-CO-NH] _n -CH₂-CO-R² (I) wherein R¹, R² and R³ have the meaning given in claim 1, is reacted. 17. Kit for the production of radiopharmaceuticals, consisting of a compound of general formula I according to one of claims 1 to 7 or a conjugate according to one of claims 12 to 14, and a reducing agent and optionally an auxiliary ligand, which in dry or in solution, and an instruction manual with a reaction Scripture to implement the connections described with technetium-99m or Re in the form of a pertech netate solution or perrhenate solution. 18. Radiopharmaceutical composition for non-inva sive in vivo representation of receptors and prescription tissue and / or atherosclerotic Plaques and / or for kidney function testing, thereby characterized that they connect after a of claims 8 to 10 or a conjugate according to ei nem of claims 12 to 14 and optionally with contains the additives common in galenics, whereby the connection in a kit according to claim 17 with Technetium-99m or Re in the form of a pertechnetate solution solution or perrhenate solution is prepared.