CA2156618A1 - Bifunctional chelators and their use in radiopharmaceuticals - Google Patents

Abstract

This invention relates to new technetium and rhenium chelates, methods for their production, and radiopharmaceu-ticals containing these compounds as well as to conjugates of these compounds with substances that accumulate selec-tively in diseased tissue, in particular, peptides and proteins. This invention further relates to the production of agents containing these compounds and their application in radiodiagnostics.

Description

-Bifunctional chelating agents, their technetium and rhenium complexes, methods for their production, and radiopharma-ceuticals containing these compounds.
This invention relates to new technetium and rhenium chelates, methods for their production, and radiopharmaceu-ticals containing these compounds as well as to conjugates of these compounds with substances that accumulate selec-tively in diseased tissue, in particular, peptides and proteins. This in~éntion further relates to the production of agents containing these compounds and their application in radiodiagnostics.

Radioactive metal ions have been in use in medical diagnostics and for therapy for a longer period of time.
For example, gamma-ray emitters such as the Tc-99m isotope lS were used for detecting tumours. ~-ray emitters such as the Re-186, Re-188, and Re-189 isotopes were applied in tumour therapy. Technetium-99m is the most frequently applied radionuclide in nuclear medicine as it is particularly suitable as an isotope for in-vivo diagnostics due to its favourable physical properties (no corpuscular radiation, physical half-life of 6 h, y-radiation 140 keV) and the low exposure to radiation resulting from them. Technetium-99m is easily gained from nuclide generators in the form of pertechnetate. It can be immediately used in this form to produce kits for clinical routine applications.

The efficiency of radionuclides in in-vivo diagnostics as well as for therapeutical purposes is dependent on the specificity and selectivity of the labelled chelates with regard to the target cell. An improvement of these properties can be achieved by coupling the chelates with biomolecules according to the "drug targeting" principle.
Antibodies, their fragments, hormones, growth factors and substrates of receptors and enzymes are the obvious substances for use as biomolecules. For example, British ' i 21S6618 -patent application GB 2,109,407 describes the use of radiolabelled monoclonal antibodies against tumour-associated antigens for in-vivo tumour diagnostics. Direct labelling of protein using technetium-99m, either via donor groups (amino, amide, thiol groups, etc.) of the protein (Rhodes, B.A. et al., J. Nucl. Med. 1986, 27, 685-693) or by introducing complexing agents (US Patent 4,479,930 and Fritzberg, A.R. et al., J. Nucl. Med. 1986, 27, 957) were described likewise. These experimental methods, however, are not available for clinical application because their selectivity is too low and their background activity is too high to facilitate in-vivo imaging.

The first receptor-binding radiopharmaceuticals were described by J. P. DiZio et al. (J. Nucl . Med. 1992, 33;
558-569 and Bioconjugate Chem. 1991, 2; 353-366). These compounds were not found suitable for in-vivo imaging. The fact that the compounds described have to be extracted from the labelling medium has proved to be a great disadvantage.
These conditions are unsatisfactory as a user should be exposed to as little radiation as possible when preparing the kit and because of the fact that only few clinics have suitable laboratories for carrying out such work.

Cyclic amines as described by Volkert et al. (Appl. Radiol.
Isot. 1982, 33; 891-896) and Troutner et al. (J. Nucl. Med.
1980, 21; 443-448) as well as N2S2 and N3S systems are per se of little use as tissue-specific radiopharmaceuticals as they lack reactive groups to combine with biomolecules or proteins (M. Borel et al., Appl.Radiat.Isot. 1992, 43, 425-436).
While pH values from 10 to 13 are required for labelling the above mentioned cyclic amines as well as N2O2 systems known from literature (Pillai, M.R.A. et al.; Inorg. Chem.
1990, 29, 1850-1856), N2S2 and N3S systems can already be complexed, as a rule, at pH values from 9 to 11. But these conditions are still too extreme for labelling unstable .2i~

-conjugates of biomolecules and macromolecules. This is all the more true as some systems such as L,L-ethylene dicysteine (L,L-EC) can only be sufficiently labelled at pH
values ~ 10 (Verbruggen, A.M. et al.; J. Nucl. Med. 1992, 33, 551-557). MAG3 as it is known from literature even requires exposure for ten minutes to temperatures from 90 to 100 C (Bannister, K.M. et al.; J. Nucl. Med. 1990, 31, 1568-1573) to be ~roduced in a radiochemical purity that is sufficient for clinical applications. These conditions are not satisfactory ~or everyday clinical routine.

N2S2 and N3S systems as described by G. Bormans et al.
(Nucl. Med. Biol. 1990, 17, 499-506) and in European Patent Specifications EP 0 173 424 and EP 0 250 013 meet the requirement of sufficient s~ability of the respective technetium-99m complexes but are discharged too rapidly and without specific accumulation in the organism. Thus they are only used clinically, though to a limited extent, in renal function diagnostics. Their use is limited mainly because the demand has increased for substances that accu-mulate specifically in diseased tissues.

To solve the refined problems of nuclear medicine, there isan increasing need for stable bifunctional complexing agents that do not show the disadvantages mentioned when complexing and are capable of forming conjugates with bio-molecules and macromolecules without considerably affectingthe specif-icity and selectivity of the latter. At the same time, all requirements regarding radiation dose, stability, and solubility have to be met to use these compounds in human beings.

It is thus a problem of this invention to provide these compounds and conjugates, to create a fairly simple method for their production, and to provide a kit formulation for clinical application of these compounds/conjugates according to the invention. The present invention solves this problem.

61~

The compounds according to the invention are characterized by the general formula (IJ

Rl\ /Y Z
CH

( ) ~M/(A3) ( I ) R6~N N~R7 .(A2) wherein (Al), (A2), and (A3) are same or different and represent the general formula O (CHR )I < (CHR9)m (CR4R5)k X

their free valences being linked arbitrarily with the respective nitrogen or sulfur atoms, and wherein s 215661X

R3 and Rs are same or different and represent a hydrogen atom, or a methyl or ethyl group, and R4 is a hydrogen atom, a branched or unbranched alkyl group containing 1 to 4 carbon atoms, the C atoms of which may optionally carry additional amino groups, N(RaRb) groups (with Ra and Rb being either same or different and repre-senting branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), hydroxy groups, thiol groups, halogens, carboxy groups, alkoxy carbonyl groups containing 1 to 20 carbon atoms, acyloxy groups containing 1 to 12 carbon atoms, amino carbonyl grou~s, sulfonyl groups, amino sulfonyl groups, or phosphorus residues, R2 and R9 are same or different and represent the same as R4, k, 1, and m are same or different and stand for the numbers 0, 1, 2, 3, or 4, and X is a hydrogen atom, a carboxy group, an alkoxy group containing 1 to 20 carbon atoms, an alkoxy carbonyl group containing 1 to 20 carbon atoms, an acyloxy group containing 1 to 20 carbon atoms, an amino carbonyl group, a sulfonyl group, an amino sulfonyl group, a phosphoric acid residue, a carboxymethyl amino carbonyl group, a p-amino-phenyl group, a p-hydroxyphenyl group, a halogen atom, a hydroxy group, an amino group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry one additional hydroxy, carboxy, or amino group), a hydrazine group, a hydrazide group, a cholesteryl oxycarbonyl methyl amino carbonyl group, a cholesteryl oxycarbonyl group, a cholesteryl oxycarbonyl methyl 6 21~661~
-oxycarbonyl group, another steroid or a derivative of an ethinyl or ethenyl steroid, a substituent of the formula zl_yl_ (CH2) i-Q-CO-where Q is an -NH- or -Q-, Zl has the same meaning as Z, yl has the same meaning as Y, and i has the same meaning as m, a substituent of the formula v - u - Ql ~-where Q1 is an -NH-, -C0-, or -O-, U is a bond, a group of the formula -(OCH2C0) h- with h=1-3, or a suitable linker for coupling with bio- or macromole-cules, and V is a hydrogen atom, a hydroxy group, an N(RaRb) group (with Ra and Rb being either same or different and repre-senting branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms,the C atoms of which may optionally carry one additional hydroxy, carboxy, or amino group), a biomolecule, or a macromolecule, and Y is an unsaturated unbranched or branched chain of up to 12 carbon atoms containing at least one double and/or triple bond which may, optionally, carry additional one or several and at any position in the chain, hydroxy, carboxy, alkoxy, amino, or substituted amido groups containing 1 to 20 carbon atoms in the alkyl and/or aryl residue, Z is a hydrogen atom, a halogen atom, a carboxy group, a hydroxy group, an alkoxy carbonyl group containing 1 to 20 carbon atoms, an acyloxy group containing 1 to 20 carbon 21~6618 atoms, an alkoxy group containing 1 to 20 carbon atoms, a cholesteryl oxycarbonyl methyl amino carbonyl group, a cholesteryl oxycarbonyl group, a cholesteryl oxycarbonyl methyl oxycarbonyl group, another steroid or a derivative S of an ethinyl or ethenyl steroid, a substituent of the formula V - U - Ql - -where Q1 is an -NH-, -C0-, or -0-, U is a bond, a group of the formula -(OCH2C=0) h- with h=l-3, or a suitable linker for coupling with bio- or macro-molecules, and `~-V is a hydrogen atom, a hydroxy group, an N(RaRb) group (with Ra and Rb being either same or different and repre-senting branched or unbranched alkyl or acyl residuescontaining 1 to 20 carbon atoms, the C atoms of which may optionally carry one additional hydroxy, carboxy, or amino group), a biomolecule, or a macromolecule, and M is an element having the atomic number 43 or 75, Rl has the same meaning as R4, R6, R7, and R8 are same or different and represent a hydro-gen atom, an alkyl group containing 1 to 4 carbon atoms, the C atoms of which may carry additional hydroxy, carboxy, or amino groups, or a group of the formula -CH2-X in which X has the above meaning and their hydrosoluble salts.

Preferred compounds according to the invention of the general formula (I) are characterized in that the residue Z
is a hydrogen atom, a steroid, an ethinyl steroid or an ethenyl steroid.

Furthermore, compounds according to the invention of general formula (I) are preferred that are characterized in that residue Z is a hydrogen atom, a steroid, an ethinyl steroid or an ethenyl steroid, and that residue X is a hydrogen atom, a halogen atom, a carboxy group, an amino group, or an amido group containing 1 to 20 carbon atoms in their alkyl and/or aryl residue.

In addition, compounds according to the invention of general formula (I? are preferred that are characterized in that residue Z is a hydrogen atom, a steroid, an ethinyl steroid or an ethenyl steroid, and that residue X is a hydrogen atom, a halogen atom, a carboxy group, an amino group, or an amido ~roup containing 1 to 20 carbon atoms in the alkyl and/or aryl residue, Y is an ethinylide group, residues Rl, R3, R6, R7, and R3 represent hydrogen atoms, and k, 1, and m each represent the number 0.
Another group of preferred compounds according to the invention of general formula (I) is characterized in that residue Z is a hydrogen atom, and residue X is a choles-teryl oxycarbonyl methyl amino carbonyl group, a choles-teryl oxycarbonyl group, a cholesteryl oxycarbonyl methyl oxycarbonyl group, another steroid or a derivative of an ethine or ethene steroid.

Furthermore, preferred compounds according to the invention of general formula (I) are characterized in that residue Z
is a hydrogen atom, residue X is a cholesteryl oxycarbonyl methyl amino carbonyl group, a cholesteryl oxycarbonyl group, a cholesteryl oxycarbonyl methyl oxycarbonyl group, another steroid or a derivative of an ethine or ethene steroid, residue Y is an ethinylide group, and that residues R1, R3, R6, R7, and R8 represent hydrogen atoms, and k, 1, and m each represent the number 0.
A third group of preferred compounds according to the invention of general formula (I) is characterized in that residue Z is a hydrogen atom, residue X is a hydrogen atom, a carboxy group, or a substituent of the formula V - U - Ql _ where S Ql is an -NH-, -CO-, or -O-, U is a bond, a group of the formula -(OCH2CO)h- with h=1-3, or a suitable linker for coupling with bio- or macromole-cules, and V is a hydrogen atom, a hydroxy group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), a biomolecule, or a macromolecule.

Other preferred compounds according to the invention of general formula (I) are characterized in that residue Z is a hydrogen atom, residue X is a hydrogen atom, a carboxy group, or a substituent of the formula V - U - Ql _ where Q1 is an -NH-, -CO-, or -O-, U is a bond, a group of the formula -(OCH2CO)h- with h=1-3, or a suitable linker for coupling with bio- or macromole-cules, and-V is a hydrogen atom, a hydroxy group, an N(RaRb) group(with Ra and Rb being either same or different and repre-senting branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), a biomolecule, or a macromolecule, that residue Y
is an ethinylide group, and that residues Rl, R3, R6, R7, and R8 represent hydrogen atoms, and k, l, and m each represent the number 0.

- lo 21S6618 A fourth group of preferred compounds according to the invention of general formula (I) is characterized in that residue Z is a hydrogen atom, and residue X is a hydrogen atom, a carboxy group, an alkoxy group containing 1 to 20 carbon atoms, an alkoxy carbonyl group containing 1 to 20 carbon atoms, an acyloxy group containing 1 to 20 carbon atoms, an amino carbonyl group, a sulfonyl group, an amino sulfonyl group, a-phosphoric acid residue, a carboxymethyl amino carbonyl group, a p-aminophenyl group, a p-hydroxy-phenyl group, a ha~ogen atom, a hydroxy group, an amino group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C
atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), a hydrazine group, or a hydrazidegroup.

In addition, compounds according to the invention of general formula (I) are characterized in that residue Z is a hydrogen atom, and residue X is a hydrogen atom, a carboxy group, an alkoxy group containing 1 to 20 carbon atoms, an alkoxy carbonyl group containing 1 to 20 carbon atoms, an acyloxy group containing 1 to 20 carbon atoms, an amino carbonyl group, a sulfonyl group, an amino sulfonyl group, a phosphoric acid residue, a carboxymethyl amino carbonyl group, a p-aminophenyl group, a p-hydroxyphenyl group, a halogen atom, a hydroxy group, an amino group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), a hydrazine group, or a hydrazide group, that residue Y is an ethinylide group, and that residues R1, R3, R6, R7, and R8 represent hydrogen atoms, and k, l, and m each represent the number 0.

The present invention further relates to compounds of the general formula (II) Rl\ /Y Z
T CH
~S N R8 (A~) (A3) ( I I ) R6~N N~R7 (A2) '~.

wherein Al, A2, A3, R1, R6, R7, R8, Y and Z have the meaning specified above and wherein T is a hydrogen atom, an acetate group, a benzoate group, a p-methoxybenzyl group, an acetamidomethyl group, a benzamidomethyl group, a trimethyl acetamidomethyl group, a hydroxy acetyl group, or another suitable sulfur protective group.

Another object of this invention are conjugates of com-pounds according to the invention of general formulae (I) and (II) with substances that accumulate selectively in tissues. The fragments are bonded amidically, imidically, or ester-like, depending on the kind of substances to be conjugated. If the substances that accumulate in the tissues are peptides, proteins, antibodies, or fragments thereof, they are bonded amidically through their amino groups; if the substances that accumulate in the tissues contain hydroxy groups, they are bonded ester-likei and if the substances that accumulate in the tissues contain aldehyde groups, they are bonded imidically.

~ 12 ~ 1~ 6618 Such substances accumulating in tissues are preferred that accumulate in diseased tissues, above all in tissues that have atherosclerotic plaques.
s Furthermore, conjugates according to the invention are preferred that are characterized in that the substances that accumulate in diseased tissue are peptides such as endothelines, partial endotheline sequences, endotheline analogues, endothe~ine derivatives, or endotheline antagonists.

Particularly preferred conjugates according to the invention are characterized in that the peptides comprise the following sequences or parts thereof:

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-tyr-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, ~ 13 21~66i8 cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-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, ~

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-trp-leu-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, N-acetyl-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, or the partial sequence his-leu-asp-ile-ile-trp or the cyclic amino acid sequences Cyclo-(Dtrp-Dasp-pro-Dval-leu), Cyclo-(Dglu-ala-alloDile-leu-Dtrp).

21~6618 _ 14 The compounds according to the invention of the general formula (I) CH

S ~ M / (A3) 6,N N ~ R7 ~(A2) are produced according to a method known to a person skilled in the art, for example, by reacting, in the presence of a reductant (such,as Sn(II) chloride or dithionide) and, optionally, an auxiliary ligand (such as sodium citrate or sodium tartrate), technetium-99m in the form of pertechnetate which is easily eluted from molyb-denum-technetium generators, with a compound of the general formula (II) Rl ~Y Z
T CH

(A') ( ~3) R6~N ~N ~ R7 (A2)--' wherein Al, A2, A3, Rl, R6, R7, R8, Y, Z, and T have the meaning specified above.

The reaction is preferably carried out in an aqueous medium at room temperature. The SH protective group is split off in situ or according to the methods known to a person skilled in the art from the relevant literature, e.g. basic hydrolysis, reductive decomposition, etc. (see, for example, "Protective Groups in Organic Synthesis", T.W.
Greene, John Wiley and Sons 1981).

Another object of the present invention are methods for the production of compo*nds according to the invention of the general formula (II) T CH

(A1) (/3) ( ll ) R6,N ~ N~R7 (A2) characterized in that, in a generally known way, a) a compound of the general formula (III) R'\ /Y Z
CH
Hal N--R8 (A~ 3) ( I I I ) 6,N N ~ R7 (A2) <
wherein Al A2, A3, Rl, R6, R7, R8, Y and Z have the meaning specified above and~ Hal represents a halogen, is reacted with a compound of the general formula T - S~ M+

wherein M+ is an alkaline metal cation and T has the meaning specified above, or, b) a compound of the general formula HN(R6)-A2-N(R7)-A3-N(R8)-Y-Z

wherein A2, A3, R6, R7, and R8 have the meaning specified above, are reacted with a compound of the general formula T - S - Al - W

wherein Al and T have the meaning given in Claim 11, and W
represents a leaving group which enables A1 to react with free amino acids.
The N-haloacetyl amides required as parent substances are produced according to methods known from the relevant literature by acylating an amino function using haloacetyl `_ 1 21S6618 halides (JACS, 1969, 90, 4508; Chem. Pharm. Bull. 1981, 29(1), 128).

The required amines of the general formula HN(R6) -A2-N(R7) -A3-N(R8) -Y-Z

are synthesized according to methods of peptide synthesis known from the relevant literature (see, for example, "The Practice of Peptide Synthesis", M. Bodanszky and A.
Bodanszky; Spring~r Verlag, 1984, and Fieser, Reagents for Organic Synthesis 10, 142). N-protected a- and ~-amino acids are coupled, according to methods known to a person skilled in the art such as a (primary or secondary) amine addition/ eliminati~on reaction, with N-unprotected organic molecules using a carbonyl compound (such as acid chloride, mixed anhydride, activated ester). After separating the amino protecting group according to methods known from literature (such as basic hydrolysis, hydrogenolysis, reductive decomposition using alkaline metals in liquid ammonium), the remaining amino acid is re-derivated in a second reaction using an N-protected a- and ~-amino acid according to methods known from the relevant literature (e.g. the carbodiimide method). The amino protecting group is separated according to the above-mentioned methods described in the literature.

The compounds of the general formula T - S - Al - W

that are also required are synthesized by converting the carboxy group(s) of respective carboxylic acids according to methods known to a person skilled in the art, for example, according to the diimide method (Fieser, Reagents for Organic Synthesis 10, 142), via a mixed or cyclic anhydride (Org. Prep. Proc. Int. 1975, 7, 215), or using an activated ester (Adv. Org. Chem. Part B, 472).

The compounds according to the invention show the desired properties to a great extent. The radionuclides required for their application are stably bound in the complex.

The coupling options of the chelating agents according to the invention are of particular interest; these agents can be viewed as being trifunctional as they facilitate coupling via a multiple bond as well as via a carboxy or amino group besides metal complexing in Al, A2, or A3.

It is of particular relevance to the invention that labelling methods be provided which permit rapid and quantitative reacti~on of the compounds of the invention under more gentle conditions than those of known methods, and immediately before use of the radiopharmaceutical.

It is of great advantage to link the chelating agent with steroids via the multiple bond as this allows both coupling to a -CH2 group of the steroid or bonding to the 17-ethinyl or 17-ethenyl group of steroids.

Some technetium-99m labelled steroid derivatives have a surprisingly strong affinity for their respective steroid hormone receptor. Thus, the technetium complex of 3,17~-dihydroxy-17a-(5-[2-benzoylthioacetyl-glycyl-glycyl]-amino-pent-1-(E)-en-3-in)-1,3,5-estratriene (Example 13b) showed a similar receptor affinity as 17~-estradiol in an in-vitro cytosol test (Carlson, K.E. et al. 1989, 32, 345-355). This makes these compounds excellently suited for re-ceptor imaging using technetium-99m and for the diagnosis and therapy of steroid-dependent tumours.
Another advantage of the present invention is the fact that, due to the option of linking either via the carboxy group or via the multiple bond, it allows to control the solubility and pharmacokinetics of the complexes by chemi-cal substitution at the level of complexing agents.

19 21~661~

Biodegradable groups or linkers in X are of particular significance here.

Compounds containing lipophilic substituents in X or Z show surprising properties, such as the compound of Example 9, as they accumulate in atherosclerotic plaques and thus provide a way to visualize atherosclerotic modifications on vessel walls.

Selection of appropriate bio- and macromolecules (e.g.
monoclonal antibod~es, steroids, ...) in X or Z yields complexes according to the invention that show a surpris-ingly great extent of tissue and organ specificity and may therefore contribute excellently to solving a number of diagnostic and therapeutical problems.

The complexing ligands obtained in this way of the general formula (II) in which Z is a hydrogen atom and/or ligands of the general formula (II) containing an ethinyl function may be coupled with iodovinyl steroids according to methods known to a person skilled in the art (e.g. R. Rossi et al., Tetrahedron 1983, 39, 287). The iodovinyl steroids required are synthesized according to methods known from the relevant literature (e.g. H. Hofmeister et al., Tetrahedron 1986, 42, 357S).

The carboxy groups of compounds of the general formula (II) may be converted, for example, according to the diimide method (Fieser, Reagents for Organic Synthesis 10, 142), via a mixed or cyclic anhydride (Org. Prep. Proc. Int.
1975, 7, 215), or using an activated ester (Adv. Org. Chem.
Part B, 472).

The chelating agents are coupled to bio- or macromolecules according to methods known to a person skilled in the art by converting the carboxy group(s) at the chelating agent, for example, according to the diimide method (Fieser, Reagents for Organic Synthesis 10, 142), via a mixed or cyclic anhydride (Org. Prep. Proc. Int. 1975, 7, 215), or using an activated ester (Adv. Org. Chem. Part B, 472), and subsequent formation of a covalent bond by reaction with a nucleophilic group of the bio- or macromolecule.

The chelating agents obtained in this way may also be linked with bio- or macromolecules that are known to accu-mulate to a particularly great extent in the organ, organic part, or tissue to be examined. Among such molecules are, for example, enzymes, hormones, polysaccharides such as dextrane or starches, porphyrins, bleomycins, insulin, prostaglandins, steroid hormones, amino sugar, amino acids, peptides such as polylysine, proteins (such as immoglobu-lins, monoclonal antibodies, lectins), lipides (including liposomes), and nucleotides of the DNA and RNA types.
Conjugates with albumins such as human serum albumin, with antibodies such as monoclonal antibodies, antibodies spe-cific to tumour-associated antigens, or antimyosin are of particular significance here. Suitable synthetic polymers such as polyethylene imines, polyamides, etc. may be linked instead of biological macromolecules.

The pharmaceuticals produced from these conjugates are suited for application in tumour diagnostics and tumour therapy, diagnosis of atherosclerosis, or receptor imaging.
Bonding affinity and specificity of the produced pharmaceu-tical to the target organ, target organ part, or targettissue should be impaired only slightly, or not at all, by forming the conjugate.

The radiopharmaceuticals of the invention are also produced in a generally known way by dissolving or suspending the complexing agents according to the invention and their conjugates in an aqueous medium, optionally by adding the adjuvants common in galenics, and subsequent optional lyophilization of the solution or suspension. Among the suitable additives are physiologically tolerable buffers (such as tromethamine), auxiliary ligands (such as sodium 21 ~156618 , citrate or sodium tartrate), reductants (such as tin (II) chloride) or, if required, electrolytes such as sodium chloride, or, if required, (one of) the adjuvants common in galenics (such as lactose, mannite) and/or surfactant(s) (such as lecithine, Tween~, Myrj~). The composition of additives used should facilitate the production of the compounds according to the invention.

Another object of the present invention is a kit for pro-ducing radiopharmaceuticals consisting of a compound of the general formula (I~), a reductant and, optionally, an auxiliary ligand, said substances being either dry or in solution, instructions for use including instructions for reacting the compounds described with technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.

The radiopharmaceuticals according to the invention are applied at doses from 0.1 mCi to 50 mCi, preferably at a dose between 0.5 mCi and 30 mCi, per 70 kg of a patient's body weight. Details of appli~ation and dosage are de-scribed, for example, in "Radiotracers for Medical Applica-tions" CRC Press, Boca Raton, Florida. The radiopharmaceu-ticals are designed for intravenous administration. For radiodiagnostic and radiotherapeutic purposes, the complex compounds/conjugates are used in the form of complexes with radionuclides of elements having the atomic numbers 43 or 75.

The radiopharmaceuticals according to the invention meet the various requirements to be appropriate for use as radiopharmaceuticals in radiodiagnostics and radiotherapy.
They are excellently suited for accumulating in target tissues after i.v. administration, thus permitting a non-invasive diagnosis of the respective tissues. Solubility in water of the radiopharmaceuticals according to the invention is ensured, if required, by adjuvants common in galenics as described above. In addition, the radiopharma-ceuticals according to the invention do not only have greatin-vitro stability but also a surprisingly great in-vivo stability; therefore, there is no, or at least no clini-cally relevant, release or exchange of the radionuclide bound in the complex.

The preferred radiopharmaceuticals according to the inven-tion are furthermore characterized in that they contain the compounds according to the invention of the general formu-lae (I) or (II) in the form of liposomes, and that the compound according to the invention of general formula (I) is optionally prepared in a kit using technetium or rhenium in the form of their permetallates.

The radiopharmaceut`icals according to the invention may also be used in radioimmuno- or radiation therapy. These differ from radiodiagnostics only in the type and quantity of radionuclide used. Its objective is to destroy tumour cells using short-wave rays of a fairly short range. Among the suitable ~-ray emitting isotopes are rhenium-186 and rhenium-188.

The radiopharmaceuticals according to the invention are suited for non-invasive in-vivo imaging of tissues containing steroid receptors, for example, steroid hormone dependent tumours.
The radiopharmaceuticals according to the invention are suited for non-invasive in-vivo imaging of atherosclerotic vessel changes, for example, of plaques.

All in all, new complexing agents and metal complexes have been successfully synthesized that open up new ways for diagnostic and therapeutic medicine. This is desirable mainly with a view to the development of novel imaging procedures for nuclear diagnostics.

_ 23 The invention shall now be explained in more detail by the following examples.

21~6618 _ Example 1 a) S-benzoyl thioacetyl glycyl glycyl propargyl amide [la]

A solution of 215.1 mg (1.22 mmol) of potassium thiobenzoate in anhydrous DMF is added by dropping, and in a nitrogen atmosphere, to a solution of 150 mg (0.61 mmol) of chloro-acetyl glycyl glycyl propargyl amide in anhydrous DMF. Then, catalytic quantities of sodium iodide are added, and the batch is heated for 2 hours to 110 C. After cooling down to room temperature, ~he reaction mixture is stirred into 1 N
HCl and extracted with acetic ester until the product is no longer contained in the aquèous phase. The organic phases are evaporated to dryness under reduced pressure, again taken up in acetic ~ster, washed with water, and dried above Mg2SO4. After drawing off the solvent under reduced pres-sure, the product is recrystallized from methanol or, if re-quired, purified by column chromatography using CH2Cl2/MeOH
(9:1).
Yield: 76~ of the title compound [la]
Cl6Hl7N304S (MW: 347.39 lH NMR (d6-DMS0):
= 3.11 (t; lH, -C-CH) 3.69 (d; 2H, -NH(CH2)CO-) 3.78 (d; 2H, -NH(CH2)C0-) 3.84 - 3.88 (m; 2H, -NH(CH2)-C-CH) 3.90 (s; 2H, -SCH2CO-) 7.55 - 7.61 (m; 2H, ArH) 7.69 - 7.74 (m; lH, ArH) 7.93 - 7.97 (m; 2H, ArH) 8.20 -(t; lH, -NH-) 8.27 (t; lH, -NH-) 8.50 (t; lH, -NH-) b) Technetium-99m complex tlb] of S-benzoyl thioacetyl glycyl glycyl propargyl amide A solution of 0.5 mg (1.44 ~mol) of S-benzoyl thioacetyl glycyl glycyl propargyl amide [la] in 50 ~l of 1 N soda lye is diluted with 250 ~l of phosphate buffer (Na2HPO4, 0.5 mol/l, pH 8.5). Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added. The reaction mixture i,s mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 mol;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

Example 2 a) 2-acetyl thiosuccinyl glycyl glycyl propargyl amide ~2a]

5.89 g of (37.5 mmol) of glycyl glycyl propargyl amide are dissolved in a nitrogen atmosphere in 60 ml of anhydrous DMF, mixed with 8.50 g (48.7 mmol) of acetyl mercaptosuc-cinic anhydride and kept agitated overnight at room temperature. The reaction mixture is then evaporated to dryness under reduced pressure. The residue is suspended in methanol, filtered off by suction, and recrystallized from water.
Yield: 61~ of the title compound [2a]
Cl3H17N3O6S (MW: 343.36) H NMR ( d6 - DMSO ) :
= 2.35 (s; 3H, -COCH3) 2.66; 2.83 (2x dd; 2H, -(CH2)COOH) 2.99 (t; lH, -C--CH) 3.70 - 3.77 (m; 4H, 2x -NH(CH2)-CO) 3.88 (dd; 2H, -NH(CH2)-C-CH) 4.35 (t; lH, S-CH(CH2COOH)-CH2-) 8.07 (t; lH, -~NH-) 8.12 (t; lH, -NH-) 8.26 (t; lH, -NH-~

b) Technetium-99m complex [lb] of 2-acetyl thiosuccinyl glycyl glycyl p~opargyl amide A solution of 0.5 mg (1.45 ~mol) of 2-acetyl thiosuccinyl glycyl glycyl propargyl amide [2a] in 50 ~l of 0.1 N soda lye is diluted with 250 ~l of phosphate buffer (Na2HPO4, 0.5 mol/l, pH 8.5). Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate sPlution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added. The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

27 21~ 6618 Example 3 a) S-acetyl thioacetyl glutaminyl glycyl propargyl amide [3a]
A solution/suspension of 2.0 g (8.3 mmol) of glutaminyl glycyl propargyl amide in 80 ml of anhydrous DMF is slowly mixed with a solution of 5.3 g (8.4 mmol) of S-acetyl mercaptoacetic N-hydroxysuccinimide ester in anhydrous THF.
The mixture is hydrolyzed after 4 hours of stirring at room temperature. Then~the solvent is removed under reduced pressure, and the residue recrystallized from methanol/water.
Yield: 45~ of the title compound [3a]
Cl4Hl9N306S (~W: 357.36) lH NMR (d6-DMSO):
~ = 1.64 - 1.92 (m; 2H, -CHCH2CH2COOH) 2.31 (s; 3H, -COCH3) 2.36 (t; 2H, -CHCH2CH2COOH) 3.02 (t; lH, -C--CH) 3.70 (s; 2H, -NH(CH2)-CO)-3.73 - 3.80 (m; lH, -CHCH2CH2COOH) 3.89 (dd; 2H, -NH(CH2)-C--CH) 8.09 (m; lH, -NH-) 8.19 (m; lH, -NH-) 8.26 (m; lH, -NH-) b) Technetium-99m complex [3b] of S-acetyl thioacetyl glutaminyl glycyl propargyl amide A solution of 0.5 mg (1.40 ~mol) of S-acetyl thioacetyl glutaminyl glycyl propargyl amide [3a] in 50 ~1 of 0.1 N
soda lye is diluted with 250 ~l of phosphate buffer (Na2HPO4, 0.5 mol/l, pH 8.5). Afterwards, 50 ~1 of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~1 of 0.2 molar tin(II) chloride dihydrate solution are added.
The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated -at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V.); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.
, Example 4 a) S-acetyl thioac~tyl glycyl glutaminyl propargyl amide ~4a]

A solution/suspension of 2.0 g (8.3 mmol) of glycyl glutaminyl propargyl amide in 80 ml of anhydrous DMF is slowly mixed with a solution of 5.3 g (8.4 mmol) of S-acetyl mercaptoacetic N-hydroxysuccinimide ester in anhydrous TH~. The mixture is hydrolyzed after 4 hours of stirring at room temperature. Then the solvent is removed under reduced pressure, and the residue recrystallized from methanol/water.
Yield: 61~ of the title compound [4a]
Cl4H1gN3O6S (MW: 357.36) 1H NMR (d6-DMSO):
~ = 1.60 - 1.88 (m; 2H, -CHCH2CH2COOH) 2.30 (s; 3H, -COCH3) 2.28 (t; 2H, -CHCH2CH2COOH) 3.04 (t; lH, -C3CH) 3.68 (s; 2H, -NH(CH2)-CO) 3.70 - 3.77 (m; lH, -CHCH2CH2COOH) 3.88 (dd; 2H, -NH(CH2)-C3CH) 4.19 (s; 2H, -SCH2CO-) 8.11 (m; lH, -NH-) 8.23 (m; lH, -NH-) _ 29 2156618 8.35 (m; lH, -NH-) b) Technetium-99m complex [4b] of S-acetyl thioacetyl glycyl glutaminyl propargyl amide A solution of 0.5 mg (1.40 ~mol) of S-acetyl thioacetyl glycyl glutaminyl propargyl amide [4a] in 50 ~l of 0.1 N
soda lye is diluted with 250 ~l of phosphate buffer (Na2HPO4, 0.5 mol/1, pH 7.5). Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added.
The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HP04; 0.01 M;
pH 2.0) 50:50 (V/V); flow ratê: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95%.
Example 5 a) S-(p-methoxybenzyl) cysteinyl glycyl glycyl propargyl amide [Sa]

9.8 g (0.02 mol) of N- tert. -butoxycarbonyl-S-(p-methoxyben-zyl) cysteinyl glycyl glycyl propargyl amide are dissolved at 0C in trifluoroacetic acid and stirred in an ice bath for 4 hours. Then the solution is evaporated to dryness under reduced pressure, the residue is mixed with ethanol and evaporated once again. The residue is purified by column chromatography above silica gel [CH2Cl2/CH3OH
(5:1)]. The product crystallizes after evaporating.
Yield: 93~ of the title compound [5a]

_ 30 2156618 C18H24N4O4S (MW: 392.48) H NMR (CD30D):
= 2.54 (t; lH, -C-CH) 2.63 - 2.86 (m; 2H, -CHCH2S-) 3.48 - 3.s2 (m; lH, -CHCH2S-) 3.71 (s; 2H, -NH(CH2)CO-) 3.77 (s; 3H, -OCH3) 3.85 (s; 2H, --NH(CH2)CO-) 3.92 (s; 2H, -CH2Ar) 3.97 (dd; 2H, .~NH(CH2)-C-CH) 6.86; 7.25 (AA'BB'; 4H, ArH) b) Technetium-99m complex of cysteinyl glycyl glycyl propargyl amide~[Sb]

A solution of 0.5 mg (1.27 ~mol) of S-(p-methoxybenzyl) cysteinyl glycyl glycyl propargyl amide [5a] in liquid HF
is stirred for 15 minutes at 0C. After evaporating the solvent at room temperature, the residue is taken up in 50 ~l of 1 N soda lye and diluted with 250 ~l of phosphate buffer (Na2HPO4, 0.5 mol/l, pH 7.5). Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added.
The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, S ~m;
gradient from 100% A to 100% B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0;01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

~ 31 2156618 Example 6 a) N-[2-acetylthio-3-cholesteryl oxycarbonyl propionyl]-glycyl glycyl propargyl amide t6a]

A solution of 5.0 g (8.9 mmol) of 3-acetylthio cholesterol succinate in anhydrous THF is cooled in an ice bath and mixed in excess with dry triethyl amine. Afterwards, 1.9 g (17.8 mmol) of ethyl chloroformate are added by dropping.
The batch is allowed to warm up to 0C, then mixed with 3.0 g (18.0 mmol) of glycyl glycyl propargyl amide and stirred at room temperature for 3 hours. The reaction mixture is hydrolyzed with water and extracted with acetic ester until the product is no longer contained in the aqueous phase. The organic phases are evaporated to dryness under reduced pressure, again taken up in acetic ester, washed with water, and dried above Mg2SO4. After drawing off the solvent under reduced pressure, the product is recrystallized from methanol or, if required, purified by column chromatography using CH2Cl2/MeOH (9:1).
Yield: 67~ of the title compoûnd [6a]
C40H61N306S (MW: 712.01) H NMR (CDC13):
= 0.68 - 2.08 (m; 41H, steroidal) 2.30 (d; 2H, -C=CHCH2- steroidal) 2.32 (s; 3H, -COCH3) 2.76 - 3.00 (m; 2H, -(CH2)COO-) 3.10 (t-; lH, -C-CH) 3.66 - 3.73 (m; 4H, 2x -NH(CH2)CO-) 3.84 (d; 2H, -NH(CH2)-C--CH) 4.39 (t; lH, -S-CH(CH2COOH)-CH2-) 4.52 - 4.63 (m; lH, -(CH)O steroidal) 5.48 (d; lH, -C=CH- steroidal) 8.05 (t; lH, -NH-) 8.11 (t; lH, -NH-) 8.23 (t; lH, -NH-) 2~6618 _ 32 b) Technetium-99m complex [6b] of N-t2-acetylthio-3-cholesteryl oxycarbonyl propionyl]-glycyl glycyl propargyl amide A solution of 0.5 mg (0.7 ~mol) of N-[2-acetylthio-3-cholesteryl oxycarbonyl propionyl]-glycyl glycyl propargyl amide [6a] in 300 ~1 of DMSO is mixed with 30 ~l of 1 N
soda lye. Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 0.4 to 0.9 mCi of pertechnetate solution from a Mo_99/Tc-99m generator are added. The reaction mixture is mixed in portions with 10 ~l of 0.2 molar tin(II) chloride dihydrate solution, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter). ~
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 30 min;
eluent A: acetonitrile/water 50:50 (V/V);
eluent B: acetonitrile; flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~-Example 7 a) Cholesterol-~N-(2-acetylthio succinyl glycyl glycyl propargyl amide-4-yl)glycine] ester ~7a]

A solution of 2.0 mg (5.8 mmol) of 2-acetylthio succinyl glycyl glycyl propargyl amide [2a] in anhydrous THF is cooled in an ice bath and mixed in excess with dry triethyl amine. Afterwards, 1.3 g (11.6 mmol) of ethyl chloroformate are added by dropping. The batch is allowed to warm up to 0C, then mixed with 5.0 g (12.0 mmol) of glycine cholesterol ester and stirred at room temperature for 3 hours. The reaction mixture is hydrolyzed with water and extracted with acetic ester until the product is no longer _ 33 21S6618 contained in the aqueous phase. The organic phases are evaporated to dryness under reduced pressure, again taken up in acetic ester, washed with water, and dried above Mg2SO4.
After drawing off the solvent under reduced pressure, the product is recrystallized from methanol or, if required, purified by column chromatography using CH2Cl2/MeOH (9:1).
Yield: 41% of the title compound [7a]
C42H64N306S (MW: 769.06) lH NMR (CD30D):
~ = 0.68 - 2.10 (m;'41H, steroidal) 2.32 (d; 2H, -C=CHCH2- steroidal) 2.33 (s; 3H, -COCH3) 2.81 - 3.10 (m; 2H, -CH(CH2)COO-) 3.08 (t; lH, -C--~H) 3.41 (d; 2H, -NH(CH2)CO-) 3.64 - 3.71 (m; 4H, 2x -NH(CH2)CO-) 3.83 (d; 2H, -NH(CH2)-C-CH) 4.35 (t; lH, -S-CH(CH2COOH)-CH2-) 4.61 - 4.72 (m; lH, -(CH)O- steroidal) 5.38 (d; lH, -C=CH- steroidal) 8.05 (t; lH, -NH-) 8.09 (t; lH, -NH-) 8.13 (t; lH, -NH-) 8.25 (t; lH, -NH-) b) Technetium-99m complex [7b] of cholesterol-tN-(2-acetylthio-succinyl glycyl glycyl propargyl a~; de-4-yl)glycine] ester A solution of 0.5 mg (0.65 ~mol) of cholesterol-N-[2-acetylthio-succinyl glycyl glycyl propargyl amide-4-yl)glycine] ester [7a] in 300 ~1 of DMSO is mixed with 30 ~1 of 0.1 N soda lye. Afterwards, 50 ~1 of 0.15 molar trisodium citrate dihydrate solution and 0.4 to 0.9 mCi of pertechnetate solution from a Mo-99/Tc-99m generator are added. The reaction mixture is mixed in portions with 10 ~l ~ 34 21~661~

of 0.2 molar tin(II) chloride dihydrate solution, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100% B within 30 min;
eluent A: acetonitrile/water 50:50 (V/V);
eluent B: acetonitrile; flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95%.

Example 8 a) N-dodecanoyl-S-(p-methoxybenzyl) cysteinyl glycyl glycyl propargyl amide [8a]

2.0 g (5.1 mmol) of S-(p-methoxybenzyl) cysteinyl glycyl glycyl propargyl amide [5a] are dissolved in CH2Cl2 and mixed with a small amount of NEt3. Afterwards, 1.2 g (5.5 mmol) of dodecanoic acid chloride in CH2Cl2 are added by dropping and stirred at room temperature for 2 hours. The mixture is hydrolyzed with water and extracted with CH2Cl2.
After drying, the product is evaporated under reduced pressure and the residue chromatographed using CH2Cl2/MeOH
(9:1) above silica gel.
Yield: 80% of the title compound [8a]
C30H46N4OsS (MW: 574.79) H NMR (d6-DMSO):
= 0.85 (t; 3H, -(CH2)8CH3) 1.19 - 1.30 (m; 16H, -(CH2)8CH3) 1.46 - 1.54 (m; 2H, -CH2-CH2CONH) 2.08 - 2.20 (m; 2H, -CH2-CH2CONH) 2.51 - 2.79 (m; 2H, -CHCH2S) 3.08 (t; lH, -C-CH) 3.74 (s; 3H, OCH3) 3.67 - 3.78 (m; 4H, 2x -NH(CH2)CO-) ~ 35 2156618 3.76 (d; 2H, -CH2Ar) 3.85 - 3.88 (m; 2H, -NH(CH2)-C_CH) 4.49 - 4.55 (m; lH, -CHCH2S-) 6.85; 7.23 (AA'BB'; 4H, ArH) 8.04 - 8.09 (m; 2H, 2x -NH-) 8.22 (m; lH, -NH-) 8.33 (m; lH, -NH-) b) Technetium-99m complex [8b] of N-dodecanoyl cysteinyl glycyl glycyl ~ropargyl amide A solution of 0.5 mg (0.9 ~mol) of N-dodecanoyl cysteinyl glycyl glycyl propargyl amide [8a] in liquid HF is stirred for 15 minutes at 0~C. After evaporating the solvent at room temperature, the residue is dissolved in 300 ~l of DMSO and mixed with 30 ~l of 1 N soda lye. Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 0.4 to 0.9 mCi of pertechnetate solution from a Mo-99/Tc-99m generator are added. The reaction mixture is mixed in portions with 10 ~l of 0.2 molar tin(II) chloride dihydrate solution, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

21~6618 Example 9 a) N-(3-hexadecyl aminocarbonyl-2-acetylthio-propionyl) glycyl glycyl propargyl amide [9a]

A solution of 2.0 g (5.8 mmol) of 2-acetylthio succinyl glycyl glycyl propargyl amide [2a] in anhydrous THF is cooled in an ice bath and mixed with 1.7 g (7.0 mmol) of hexadecylamine. Afterwards, 1.47 g (7.0 mmol) of dicyclo-hexyl carbodiimidefin anhydrous THF mixed with 2 ml of triethyl amine are added by dropping at 0C. The batch is allowed to warm up to room temperature and stirred for 3 hours at this temperature. The reaction mixture is mixed with a few drops of~acetic acid, hydrolyzed with water and extracted with acetic ester until the product is no longer contained in the aqueous phase. The organic phases are evaporated to dryness under reduced pressure, again taken up in acetic ester, washed with water, and dried above Mg2SO4.
After drawing off the solvent under reduced pressure, the product is recrystallized from methanol or, if required, purified by column chromatography using CH2Cl2/MeOH (9:1).
Yield: 69~ of the title compound ~9a]
C29H50N4OsS (MW: 566.81) H NMR (d6-DMSO):
= 0.87 (t; 3H, -CH2CH3) 1 16 - 1.35 (m; 28H, -(CH2)l4CH3) 2.33 (s; 3H, -COCH3) 2.73 - 3.10 (m; 4H, -(CH2)-) 3.07 (t; lH, -C--CH) 3.67 - 3.76 (m; 4H, 2x -NH(CH2)CO-) 3.85 - 3.89 (m; 2H, -NH(CH2)-C-=CH) 4.29 - 4.35 (m; lH, -S-CH(CH2COOH)-CH2-) 7.99 (t; lH, -NH-) 8.07 (t; lH, -NH-) 8.15 - 8.23 (m; 2H, 2x -NH-) _ 37 21S6618 b) Technetium-99m complex [9b] of N-(3-hexadecyl aminocarbonyl-2-acetylthio-propionyl) glycyl glycyl propargyl amide A solution of 0.5 mg (0.9 ~mol) of N-(3-hexadecyl aminocarbonyl-2-acetylthio-propionyl) glycyl glycyl propargyl amide[9a] in 300 ~l of DMSO is mixed with 30 ~l of 1 N soda lye. Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 0.4 to 0.9 mCi of pertechne-tate solution from~a Mo-99/Tc-99m generator are added. The reaction mixture is mixed in portions with 10 ~l of 0.2 molar tin(II) chloride dihydrate solution, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter). ~
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.
Example 10 a) N-dodecanoyl homocysteinyl glycyl glycyl propargyl amide ~lOa]

A solution of 2.0 g (6.7 mmol) of N-dodecanoyl homocysteinyl thiolactone in anhydrous THF is mixed with a solution of 1.2 g (7.0 mmol) glycyl glycyl propargyl amide in DMF. After adding 1 ml of triethyl amine, the batch is refluxed for 3 hours. It is then evaporated under reduced pressure, the residue is taken up in CH2Cl2 and washed with diluted HCl. The organic phases are washed neutrally with water and dried above Mg2SO4. After evaporating the solvent under reduced pressure, the product is recrystallized from 38 21~ 66 18 methanol and, optionally, purified by column chromatography using CH2Cl2/MeOH (9:1).
Yield: 58~ of the title compound [lOa]
C23H40N404S (MW: 468.66) lH NMR (d6-DMSO):
= 0.86 (t; 3H, -(CH2)8CH3) 1.20 - 1.33 (m; 16H, -(CH2)8CH3) 1.48 - 1.57 (m; 2H, -CH2-CH2CONH-) 2.10 - 2.19 (m;j 2H, -CH2-CH2CONH-) 2.56 - 2.73 (m, 2H, -CHCH2CH2SH) 3.10 (t; lH, -C-CH) 3.38 - 3.44 (m; 2H, -CHCH2CH2SH) 3.52 (s; lH, -SH) 3.65 - 3.73 (m; 4H, 2x -NH(CH2)CO-) 3.86 - 3.90 (m; 2H, -NH(CH2)-C--CH) 4.48 - 4.54 (m; lH, -CHCH2CH2SH) 8.05 - 8.10 (m; 2H, 2x -NH-) 8.24 (t; lH, -NH-) 8.35 (t; lH, -NH-) b) Technetium-99m complex tlOb] of N-dodecanoyl homocysteinyl glycyl glycyl propargyl amide A solution of 0.5 mg (1.06 ~mol) of N-dodecanoyl homocys-teinyl glycyl glycyl propargyl amide [lOa] in 300 ~l of DMSO is mixed with 30 ~l of 1 N soda lye. Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 0.4 to 0.9 mCi of pertechnetate solution from a Mo-99/Tc-99m generator are added. The reaction mixture is mixed in portions with 10 ~l of 0.2 molar tin(II) chloride dihydrate solution, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4i 0.01 M; pH 2.0);

~ 39 2156618 eluent B: acetonitrile/phosphate buffer (Na2HP04; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

Ex~mple 11 a) N-decanoyl homocysteinyl glycyl glycyl propargyl amide [lla]

A solution of 2.0 g (7.4 mmol) of N-decanoyl homocysteinyl thiolactone in anhydrous THF is mixed with a solution of 1.35 g (8.0 mmol) ~f glycyl glycyl propargyl amide in DMF.
After adding 1 ml of triethyl amine, the batch is refluxed for 3 hours. It is then evaporated under reduced pressure, the residue is taken up in CH2Cl2 and washed with diluted HCl. The organic phases are washed neutrally with water and dried above Mg2SO4. After evaporating the solvent under reduced pressure, the product is recrystallized from methanol and, optionally, purified by column chromatography using CH2Cl2/MeOH (9:1).
Yield: 67~ of the title compound [lla]
C21H36N404S (MW: 440.61) H NMR (d6-DMSO):
= 0.85 (t; 3H, -(CH2)6CH3) 1.18 - 1.30 (m; 12H, -(CH2)6CH3) 1.45 - 1.53 (m; 2H, -CH2-CH2CONH-) 2.11 - 2.22 (m; 2H, -CH2-CH2CONH-) 2.55 - 2.70 (m; 2H, -CHCH2CH2SH) 3.09 (t; lH, -CaCH) 3.37 - 3.42 (m; 2H, -CHCH2CH2SH) 3.50 (s; lH, -SH) - 3.65 - 3.75 (m; 4H, 2x -NH(CH2)CO-) 3.87 - 3.92 (m; 2H, -NH(CH2)-C_CH) 4.48 - 4.53 (m; lH, -CHCH2CH2SH) 8.08 - 8.12 (m; 2H, 2x -NH-) ~ 40 2156618 8.22 (t; lH, -NH-) 8.32 (t; lH, -NH-) b) Technetium-99m complex [llb] of N-decanoyl homocysteinyl glycyl glycyl propargyl amide A solution of 0.5 mg (1.1 ~mol) of N-decanoyl homocysteinyl glycyl glycyl pro~argyl amide [lla] in 300 ~1 of DMSO is mixed with 30 ~l of 1 N soda lye. Afterwards, 50 ~1 of 0.15 molar trisodium citrate dihydrate solution and 0.4 to 0.9 mCi of pertechnetate solution from a Mo-99/Tc-99m generator are added. The reaction mixture is mixed in portions with 10 ~l of 0.2 molar tin(II) chloride dihydrate solution, incubated at room t~e~mperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95% .

Example 12 a) N-hexanoyl homocysteinyl glycyl glycyl propargyl amide [12a]

A solution of 2.0 g (9.3 mmol) of N-hexanoyl homocysteinyl thiolactone in anhydrous THF is mixed with a solution of 1.7 g (10.0 mmol) of glycyl glycyl propargyl amide in DMF.
After adding 1 ml of triethyl amine, the batch is refluxed for 3 hours. It is then evaporated under reduced pressure, the residue is taken up in CH2Cl2 and washed with diluted HCl. The organic phases are washed neutrally with water and dried above Mg2SO4. After evaporating the solvent under - 21~6618 reduced pressure, the product is recrystallized from methanol and, optionally, purified by column chromatography using CH2Cl2/MeOH (9:1).
Yield: 60~ of the title compound [12a]
5C17H2sN44S (MW: 384.50) H NMR (d6-DMSO):
= 0.86 (t; 3H, -(CH2)2CH3) 1.20 - 1.31 (m; 12H, -(CH2)2CH3) 1.47 - 1.54 (m; 2H, -CH2-CH2CONH-) 2.10 - 2.19 (m;' 2H, -CH2-CH2CONH-) 2.56 - 2.68 (m; 2H, -CHCH2CH2SH) 3.11 (t; lH, -C--CH) 3.36 - 3.40 (m; 2H, -CHCH2CH2SH) 3.51 (s; lH, -SH) 3.67 - 3.76 (m; 4H, 2x -NH(CH2)CO-) 3.85 - 3.89 (m; 2H, -NH(CH2)-C--CH) 4.47 - 4.54 (m; lH, -CHCH2CH2SH) 8.03 - 8.10 (m; 2H, 2x -NH-) 8.20 (t; lH, -NH-) 8.29 (t; lH, -NH-) b) Technetium-99m complex tl2b] of N-h~yAn~yl homocysteinyl glycyl glycyl propargyl amide A solution of 0.5 mg (1.3 ~mol) of N-decanoyl homocysteinyl glycyl glycyl propargyl amide [lla] in 300 ~l of DMSO is mixed with 30 ~l of 1 N soda lye. Afterwards, 50 ~1 of 0.15 molar trisodium citrate dihydrate solution and 0.4 to 0.9 mCi of pertechnetate solution from a Mo-99/Tc-99m generator are added. The reaction mixture is mixed in portions with 10 ~l of 0.2 molar tin(II) chloride dihydrate solution, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);

eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

Example 13 a) 3,17~-dihydroxy-17a-[5-(2-benzoyl thioacetyl glycyl glycyl) amino pent-l-(E)-en-3-in]-1,3,5-estratriene [13a]
A suspension of 120.0 mg (0.24 mmol) of 17~-hydroxy-17a-iodovinyl-1,3,5-est~atriene-3-tetrahydropyranyl ether, 0.3 g (0.85 mmol) of S-benzoyl thioacetyl glycyl glycyl propargyl amide [la], 10.0 mg (0.044 mmol) of benzyl triethyl ammonium chloride, 10.6 mg (9.2 ~mol) of tetrakis(triphenyl phosphine)palladium(0), and 8.15 mg (0.043 mmol) of copper(I) iodide in 4 ml of toluene is stirred at room temperature for 72 hours. Then the mixture is mixed with water, extracted twice with toluene, and dried above Mg2SO4. After evaporating the solvent under reduced pressure, the residue is taken up in 5 ml of THF, mixed with 150 mg (0.6 mmol) of pyridine toluene-p-sulfonic acid in 5 ml of ethanol, and refluxed for 3 hours. Then the solvent is evaporated under reduced pressure, and the residue is purified by column chromatography using CH2Cl2/MeOH (8:1).
Yield: 41~ of the title compound [13a]
C36H4lN3O6S (MW: 643.80) H NMR (d6-DMSO):
~ = 0.86 (t; 3H, -CH3 steroidal) 1.15 - 2.30 (m; 13H, steroidal) 2.65 - 2.74 (m; 2H, -CH2- steroidal) 3.72 (d; 2H, -NH(CH2)CO-) 3.81 (d; 2H, -NH(CH2)CO-) 3.90 (s; 2H, -SCH2CO-) 215661~

4.02 (d; 2H, -NH(CH2)-C-C-) 5.65; 6.35 (AB; 2H, -CH=CH-) 6.43 (d; lH, steroidal) 6.50 (dd; lH, steroidal) 7.00 (d; lH, steroidal) 7.44 - 7.49 (m; 2H, ArH) 7.51 - 7.56 (m; 2H, ArH) 7.90 - 7.96 (m; 2H, ArH) 8.23 (t; lH, -NH-) 8.27 (t; lH, -NH-) 8.80 (t; lH, -NH-) b) Technetium-99m complex ~13b] of 3,17~-dihydroxy-17a-[5-(2-benzoyl thioà-~etyl glycyl glycyl) amino pent-1-(E)-en-3-in]-1,3,5-estratriene A solution of 0.5 mg (0.8 ~mol) of 3,17~-dihydroxy-17a-[5-(2-benzoyl thioacetyl glycyl glycyl) amino pent-1-(E)-en-3-in]-1,3,5-estratriene [13a] in 250 ~l of ethanol is mixed with 50 ~l of 1 N soda lye and incubated at room temperature for 15 minutes. Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added. The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
HAMILTON PRP-1 column, 125 x 4.6 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: acetonitrile;
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.001 mol; pH 7.4) 50:50 (V/V); flow rate: 2.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

Example 14 a) 17~-hydroxy-17a-[5-(2-benzoyl thioacetyl glycyl glycyl) amino pent-1-(E,Z)-en-3-in]-1,3,5-estrene-3-on [14a]
A suspension of 120.0 mg (0.28 mmol) of 17~-hydroxy-17a-iodovinyl-4-estrene-3-on, 0.3 g (0.85 mmol) of S-benzoyl thioacetyl glycyl glycyl propargyl amide [la], 10.0 mg (0.044 mmol) of benzyl triethyl ammonium chloride, 10.6 mg (9.2 ~mol) of tetr~kis(triphenyl phosphine)palladium(0), and 8.15 mg (0.043 mmol) of copper(I) iodide in 4 ml of toluene is stirred at room temperature for 72 hours. Then the mixture is mixed with water, extracted twice with toluene, and dried above Mg2SO4. After evaporating the solvent under reduced pressure, the residue is taken up in 5 ml of THF, mixed with 150 mg (0.6 mmol) of pyridine toluene-p-sulfonic acid in 5 ml of ethanol, and refluxed for 3 hours. Then the solvent is evaporated under reduced pressure, and the residue is purified by column chromatography using CH2Cl2/MeOH (8:1).
Yield: 39~ of the title compound [14a]
C36H43N3O6S (MW: 645.80) H NMR (d6-DMSO):
= 0.95 (t; 3H, -CH3 steroidal) 0.82 - 2.50 (m; 20H, steroidal) 3.68 (d; 2H, -NH(CH2)CO-) 3.80 (d; 2H, -NH(CH2)CO-) 3.87 (s; 2H, -SCH2CO-) 3.94 - 3.98 (m; 2H, -NH(CH2)-C--C-) 5.60; 5.95 (AB; 2H, -CH=CH-) 5.82 (s; lH, steroidal) 7.41 - 7.45 (m; 2H, ArH) 7.49 - 7.54 (m; 2H, ArH) 7.85 - 7.90 (m; 2H, ArH) 8.24 (t; lH, -NH-) 8.29 (t; lH, -NH-) 8.78 (t; lH, -NH-) ~ 45 21~6618 b) Technetium-99m complex [14b] of 17~-hydroxy-17a-[5-(2-benzoyl thioacetyl glycyl glycyl) amino pent-1-(E,Z)-en-3-in]-4-estrene-3-on A solution of 0.5 mg (0.8 ~mol) of 17~-hydroxy-17a-[5-(2-benzoyl thioacetyl glycyl glycyl) amino pent-1-(E,Z)-en-3-in]-4-estrene-3-on [14a] in 250 ~l of ethanol is mixed with 50 ~l of iN soda lye and incubated at room temperature for 15 minutes. Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added. The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m~generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
HAMILTON PRP-1 column, 125 x 4.6 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: acetonitrile;
eluent B: acetonitrile/phosph~te buffer (Na2HPO4; 0.001 mol; pH 7.4) 50:50 (V/V); flow rate: 2.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~ .

Example 15 a) S-acetyl thioacetyl sarcosyl glycyl propargyl amide [lSa]
A solution of 1.54 g (8.4 mmol) of sarcosyl glycyl propargyl amide in 30 ml of anhydrous THF is slowly mixed with a solution of 5.3 g (8.4 mmol) of S-acetyl mercaptoacetic acid N-hydroxy succinimide ester in anhydrous THF. The mixture is stirred at room temperature for 4 hours and then heated for 30 minutes to 40C. After cooling, the solvent is evaporated under reduced pressure, and the residue is purified by column chromatography above ~ 46 2 1~ 6618 silica gel [CH2Cl2/CH3OH (9:1)]. The product crystallizes from ethanol.
Yield: 88~ of the title compound [15a]
Cl2Hl7N3O4S (MW: 299.35 lH NMR (d6-DMSO):
= 2.34 (s; 3H, -COCH3) 2.81 (s; 3H, -N-CH3) 3.07 (t; lH, -C--CH) 3.70 (d; 2H, -NH(CH2)CO-) 3.82 (s; 2H, -~H(CH2)CO-) 3.86 - 3.90 (m; 2H, -NH(CH2)-C-CH) 3.93 (s; 2H, SCH2CO-) 8.13 (t; lH, -NH-) 8.22 (t; lH, -NH-) b) Technetium-99m complex tl5b] of S-acetyl thioacetyl sarcosyl glycyl propargyl amide A solution of 0.5 mg (1.67 ~mol) of S-acetyl thioacetyl sarcosyl glycyl propargyl amide [15a] in 50 ~1 of 0.1 N
soda lye is diluted with 250 ~l of phosphate buffer (Na2HPO4, 0.5 mol/l, pH 8.5). Afterwards, 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added.
The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~.

Example 16 a) 2-(S-acetylthio)succinyl sarcosyl glycyl propargyl amide [16a]
8.89 g (48.5 mmol) of sarcosyl glycyl propargyl amide are dissolved in a nitrogen atmosphere in 60 ml of anhydrous DMF, mixed with 8.5 g (48.7 mmol) of acetyl mercaptosuccin-anhydride and stirred for 1 hour at 40C. The product is precipitated with ether after cooling and centrifuged off.
The crystalline product is recrystallized from THF.
Yield: 90~ of the title compound [16a]
Cl4HlgN3O6S (MW: 357.39) H NMR (d6-DMSO):
= 2.34 (s; 3H, -CO~H3) 2.78 (s; 3H, -N-CH3) 2.70 - 3.04 (m; 2H, -(CH2)COOH) 3.07 (t; lH, -C--CH) 3.68 - 3.73 (m; 4H, 2x -NH(CH2)CO-) 3.85 - 3.89 (m; 2H, -NH(CH2)-C-CH) 4.33 - 4.38 (m; lH, -S-CH~CH2COOH)-CH2-) 8.12 (t; lH, -NH-) 8.23 (t; lH, -NH-) 12.68 (broad; lH, -COOH) b) Technetium-99m complex [16b] o~ 2-(S-acetylthio) succinyl sarcosyl glycyl propargyl amide A solution of 0.5 mg (1.4 ~mol) of 2-(S-acetylthio) succinyl sarcosyl glycyl propargyl amide [16a] in 50 ~1 of 0.1 N soda lye is diluted with 250 ~1 of phosphate buffer (Na2HPO4, 0.5 mol/l, pH 8.5). Afterwards, 50 ~1 of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~1 of 0.2 molar tin(II) chloride dihydrate solution are added.
The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).

21~6618 _ 48 Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100~ A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 95~ .

Example 17 a) (2-(S-acetylthio)succinyl sarcosyl glycyl propargyl amide-4-yl)-cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp tl7a]

A solution of 50 mg of 2-(S-acetylthio)succinyl sarcosyl glycyl propargyl amide [16a] and 15 mg of N-hydroxy suc-cinimide in anhydrous, freshly distilled DMF is cooled down to -15C and mixed with 28 mg-of dicyclohexyl carbodiimide in anhydrous DMF. The reaction mixture is stirred for 2 hours at -5C, then 2 hours at room temperature, and then cooled down to -15C. Precipitated N,N'-dicyclohexyl urea is filtered off. The filtrate is mixed with a solution of 1 mg of cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp (endotheline 1) in anhydrous DMF and stirred for 20 hours at room temperature.
The reaction mixture is evaporated under reduced pressure at room temperature. A flocculent precipitate emerges after adding diethyl ether by dropping. The precipitate is cen-trifuged and purified by preparative HPLC (gradient: aceto-nitrile/phosphate buffer). When the buffered eluate is neu-tralized, the organic solvent portion is blown off usingnitrogen, and the residue is lyophilized. The crystalline product is kept in a protective gas atmosphere (Ar).
MW: calc.: 2831.3 det.: 2831.6 (F~3-MS) 49 21~6618 b) Technetium-99m complex tl7b] of (2-(S-acetylthio) succinyl sarcosyl glycyl propargyl amide-4-yl)-cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp A solution of 0.5 mg of 2-(S-acetylthio) succinyl sarcosyl glycyl propargyl amide-4-yl)-cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp [17a] in 50 ~l of 0.1 N soda lye is diluted with 250 ~l of phosphate buffer (~a2HPO4, 0.5 mol/l, pH 8.5). Afterwards, 50 ~1 of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~1 of 0.2 molar tin(II) chloride dihydrate solution are added. The reaction mixture is mixed with a pertechnetate solut~on (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for lO minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x ~ mm, 5 ~m;
gradient from 100% A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 80~.

- Example 18 a) Cyclo(trp-leu-val-pro-asp)-cys-gly-gly-propargyl amide tl8a]
A solution of 133.0 mg of cyclo(trp-leu-val-pro-asp) and 41.3 mg of hydroxy benzotriazole in 6 ml of anhydrous, freshly distilled DMF is cooled down to -15C and mixed with a solution of 51.7 mg of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride in 8 ml of DMF. The reaction mixture is stirred for 2 hours at -5C and another -- so 21~6618 2 hours at room temperature. Then, a solution of 134.0 mg of S-(p-methoxybenzyl) cysteinyl glycyl glycyl propargyl amide [5a] in anhydrous DMF is slowly added by dropping.
After 7 more hours of stirring, the solution is evaporated under reduced pressure, and the peptide precipitated with ether. The protecting groups present are split off by stirring in liquid HF. After evaporating the HF, the residue is purified by preparative HPLC (gradient:
acetonitrile/phosph~ate buffer). When the buffered eluate is neutralized, the organic solvent portion is blown off using nitrogen, and the residue is lyophilized. The crystalline product is kept in a protective gas atmosphere (Ar).
MW: calc.: 865.0 det.: 865.2 (FAB-MS) b) Technetium-99m complex [18b] of cyclo(trp-leu-val-pro-asp)-cys-gly-gly-propargyl amide A solution of 0.5 mg of cyclo(trp-leu-val-pro-asp)-cys-gly-gly-propargyl amide[18a] in 300 ~l of phosphate buffer (Na2HPO4, 0.5 mol/l, pH 8.5) is mixed with 50 ~l of 0.15 molar trisodium citrate dihydrate solution and 2.5 ~l of 0.2 molar tin(II) chloride dihydrate solution are added.
The reaction mixture is mixed with a pertechnetate solution (0.4 to 0.9 mCi) from a Mo-99/Tc-99m generator, incubated at room temperature for 10 minutes, and filtered (0.2 ~m filter).
Labelling is analyzed using HPLC:
MERCK nucleosile column, 125 x 4 mm, 5 ~m;
gradient from 100% A to 100~ B within 7.5 min;
eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0);
eluent B: acetonitrile/phosphate buffer (Na2HPO4; 0.01 M;
pH 2.0) 50:50 (V/V); flow rate: 1.0 ml/min.
Radiochemical purity of the Tc-99m complex is more than 90~ .

~~ 51 21~661~

Example 19 Accumulation of N-(3-hexadecyl aminocarbonyl-2-acetyl thiopropionyl) glycyl glycyl propargyl amide, technetium-99m complex, in atherosclerotic vascular lesions of WHHLrabbits N-(3-hexadecyl ami~ocarbonyl-2-acetyl thiopropionyl) glycyl glycyl propargyl amide (produced according to Example 9a) is labelled as described in Example 9b.

99.9 GBq (2.7 mCi) of the substance labelled according to Example 9b were dilù'ted to l ml with phosphor-buffered saline and administered via the ear vein to a narcotized WHHL rabbit, Rompun/Ketavet (1:2). The rabbit was killed 5 hours after the application, and an autoradiogram as well as a Sudan(III) staining were carried out to visualize the atherosclerotic plaques (Figure 1). The accumulation factor between normal and atherosclerotic walls was between 3 and 8 depending on the thickness of the plaques (Sudan(III) staining).

Claims (19)

Claims

1. Compounds of the general formula (I) wherein (A1), (A2), and (A3) are same or different and represent the general formula their free valences being linked arbitrarily with the respective nitrogen or sulfur atoms, and wherein R3 and R5 are same or different and represent a hydrogen atom, or a methyl or ethyl group, and

2 R4 is a hydrogen atom, a branched or unbranched alkyl group containing 1 to 4 carbon atoms, the C atoms of which may optionally carry additional amino groups, N(RaRb) groups (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms,the C
atoms of which may optionally carry one additional hydroxy, carboxy, or amino group), hydroxy groups, thiol groups, halogens, carboxy groups, alkoxy carbonyl groups-containing 1 to 20 carbon atoms, acyloxy groups containing 1 to 12 carbon atoms, amino carbonyl groups, sulfonyl groups, amino sulfonyl groups, or phosphoric acid residues, R2 and R9 are same or different and represent the same as R4, k, l, and m are same or different and stand for the numbers 0, 1, 2, 3, or 4, and X is a hydrogen atom, a carboxy group, an alkoxy group containing 1 to 20 carbon atoms, an alkoxy carbonyl group containing 1 to 20 carbon atoms, an acyloxy group containing 1 to 20 carbon atoms, an amino carbonyl group, a sulfonyl group, an amino sulfonyl group, a phosphoric acid residue, a carboxymethyl amino carbonyl group, a p-aminophenyl group, a p-hydroxyphenyl group, a halogen atom, a hydroxy group, an amino group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms/the C
atoms of which may optionally carry one additional hydroxy, carboxy, or amino group), a hydrazine group, a hydrazide group, a cholesteryl oxycarbonyl methyl amino carbonyl group, a cholesteryl oxycarbonyl group, a cholesteryl oxycarbonyl methyl oxycarbonyl group, another steroid or a derivative of an ethinyl or ethenyl steroid, a substituent of the formula Z1-Y1-(CH2)i-Q-CO-where Q is an -NH- or -O-, Z1 has the same meaning as Z, Y1 has the same meaning as Y, and i has the same meaning as m, a substituent of the formula where Q1 is an -NH-, -CO-, or -O-, U is a bond, a group of the formula -(OCH2CO)h- with h=1-3, or a suitable linker for coupling with bio- or macromolecules, and V is a hydrogen atom, a hydroxy group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry one additional hydroxy, carboxy, or amino group), a biomolecule, or a macromolecule, and Y is an unsaturated unbranched or branched chain of up to 12 carbon atoms containing at least one double and/or triple bond which may, optionally, carry additional one or several and at any position in the chain, hydroxy, carboxy, alkoxy, amino, or substituted amido groups containing 1 to 20 carbon atoms in the alkyl and/or aryl residue, Z is a hydrogen atom, a halogen atom, a carboxy group, a hydroxy group, an alkoxy carbonyl group containing 1 to 20 carbon atoms, an acyloxy group containing 1 to 20 carbon atoms, an alkoxy group containing 1 to 20 carbon atoms, a cholesteryl oxycarbonyl methyl amino carbonyl group, a cholesteryl oxycarbonyl group, a cholesteryl oxycarbonyl methyl oxycarbonyl group, another steroid or a derivative of an ethinyl or ethenyl steroid, a substituent of the formula where -Q1 is an -NH-, -CO-, or -O-, U is a bond, a group of the formula -(OCH2CO)h- with h=1-3, or a suitable linker for coupling with bio- or macromolecules, and V is a hydrogen atom, a hydroxy group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), a biomolecule, or a macromolecule, and M is an element having the atomic number 43 or 75, R1 has the same meaning as R4, R6, R7, and R8 are same or different and represent a hydrogen atom, an alkyl group containing 1 to 4 carbon atoms, the C atoms of which may carry additional hydroxy, carboxy, or amino groups, or a group of the formula -CH2-X in which X has the above meaning and their hydrosoluble salts.

Compounds according to Claim 1, characterized in that Z
is a hydrogen atom, a steroid, an ethinyl steroid, or an ethenyl steroid.

3. Compounds according to Claim 2, characterized in that X
is a hydrogen atom, a halogen atom, a carboxy group, an amino group or an amido group containing 1 to 20 carbon atoms in their alkyl and/or aryl residue.

4. Compounds according to Claim 3, characterized in that Y
is an ethinyliden group, R1, R3, R6, R7, and R8 are hydrogen atoms, and k, 1, and m represent the number 0.

5. Compounds according to Claim 1, characterized-in that Z
is a hydrogen atom and X is a cholesteryl oxycarbonyl methyl amino carbonyl group, a cholesteryl oxycarbonyl group, a cholesteryl oxycarbonyl methyl oxycarbonyl group, another steroid or a derivative of an ethinyl or ethenyl steroid.

6. Compounds according to Claim 5, characterized in that Y
is an ethinyliden group, that R1, R3, R6, R7, and R8 are hydrogen atoms, and k, 1, and m represent the number 0 or 1.

7. Compounds according to Claim 1, characterized in that residue Z is a hydrogen atom, residue X is a hydrogen atom, a carboxy group, or a substituent of the formula where Q1 is an -NH-, -CO-, or -O-, U is a bond, a group of the formula -(OCH2CO)h- with h=1-3, or a suitable linker for coupling with bio- or macromolecules, and V is a hydrogen atom, a hydroxy group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), a biomolecule, or a macromolecule.

8. Compounds according to Claim 7, characterized in that Y
is an ethinyliden group, that R1, R3, R6, R7, and R8 are hydrogen atoms, and k, 1, and m represent the numbers 0, 1, or 2.

9. Compounds according to Claim 1, characterized in that Z
is a hydrogen atom, and residue X is a hydrogen atom, a carboxy group, an alkoxy group containing 1 to 20 carbon atoms, an alkoxy carbonyl group containing 1 to 20 carbon atoms, an acyloxy group containing 1 to 20 carbon atoms, an amino carbonyl group, a sulfonyl group, an amino sulfonyl group, a phosphoric acid residue, a carboxymethyl amino carbonyl group, a p-aminophenyl group, a p-hydroxyphenyl group, a halogen atom, a hydroxy group, an amino group, an N(RaRb) group (with Ra and Rb being either same or different and representing branched or unbranched alkyl or acyl residues containing 1 to 20 carbon atoms, the C atoms of which may optionally carry an additional hydroxy, carboxy, or amino group), a hydrazine group, or a hydrazide group.

10. Compounds according to Claim 9, characterized in that Y
is an ethinyliden group, that R1, R3, R6, R7, and R8 are hydrogen atoms, and k, 1, and m represent the numbers 0, 1, or 2.

11. Compounds of the general formula ( I I ) wherein A1, A2, A3, R1, R6, R7, R8, Y and Z have the meaning specified in Claim 1, and where T is a hydrogen atom, an acetate group, a benzoate group, a p-methoxybenzyl group, an acetamidomethyl group, a benzamidomethyl group, a trimethyl acetamidomethyl group, a hydroxy acetyl group, or another suitable sulfur protecting group.

12. Conjugates containing compounds of the general formulae (I) and/or (II) and substances that accumulate selectively in diseased tissues, with a covalent bond existing between these substances, said bond being amidic if the substances are amino groups such as peptides, proteins, antibodies, or their fragments, ester-like if the substances contain hydroxy groups, such as fatty alcohols, and imidic if the substances contain aldehyde groups.

13. Conjugates according to Claim 12, characterized in that the substances that accumulate in diseased tissue are peptides, in particular, endothelines, partial endo-theline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists.

14. Conjugates according to Claim 12, characterized in that the peptides contain the following sequences or parts thereof:

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-trp-leu-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, N-acetyl-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, or the partial sequence his-leu-asp-ile-ile-trp or the cyclic amino acid sequences Cyclo-(Dtrp-Dasp-pro-Dval-leu), Cyclo-(Dglu-ala-alloDile-leu-Dtrp).

15. Method for the production of compounds of the general formula (I), characterized in that technetium-99m or rhenium in the form of pertechnetate or perrhenate are reacted, in the presence of a reductant and, optionally, an auxiliary ligand, with a compound of the general formula (II) ( I I ) where A1, A2, A3, R1, R6, R7, R8, Y and Z have the meaning specified in Claim 1 and T has the meaning specified in Claim 11.

16. Method for the production of compounds of the general formula (II) ( I I ) characterized in that a) a compound of the general formula (III) ( I I I ) wherein A1, A2, A3, R1, R6, R7, R8, Y and Z have the meaning specified in claim 1 and Hal represents a halogen, is reacted with a compound of the general formula T - S- M+

wherein M+ is an alkaline metal cation and T has the meaning specified in claim 11, or, b) a compound of the general formula HN(R6)-A2-N(R7)-A3-N(R8)-Y-Z

wherein A2, A3, R6, R7, and R3 have the meaning specified in Claim 1, are reacted with a compound of the general formula wherein A1 and T have the meaning given in Claim 11, and W represents a leaving group which enables A1 to react with free amino acids.

17. Kit for producing radiopharmaceuticals consisting of a compound of the general formula (II) according to Claim 11, a reductant and, optionally, an auxiliary ligand, said substances being either dry or in solution, instructions for use including instructions for reacting the compounds described with technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.

18. Radiopharmaceutical formulation for non-invasive in-vivo visualization of receptors and tissue containing receptors and/or atherosclerotic plaques, characterized in that it contains a compound according to Claim 1, optionally with the adjuvants common in galenics, and that the compound according to Claim 1 is prepared in a kit according to Claim 17 using technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.

19. Radiopharmaceutical formulation according to Claim 18, characterized in that it contains a compound according to Claim 1 in the form of liposomes, and that the compound according to Claim 1 is prepared in a kit according to Claim 17 using technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution.