AU666059B2 - New agents for diagnosis of vascular diseases - Google Patents

Abstract

The present invention relates to metal complexes and metal complex conjugates of endothelins, endothelin derivatives, partial sequences of endothelins, endothelin analogues or endothelin antagonists, iodinated endothelin derivatives, partial sequences of endothelins, endothelin analogues or endothelin antagonists, and to compositions containing these compounds, to their preparation as diagnostic agents, and to processes for the preparation of these compounds and compositions. Used for this purpose are metal complexes and metal complex conjugates of endothelins, endothelin derivatives, partial sequences of endothelins, endothelin analogues or endothelin antagonists, as well as iodinated endothelin derivatives, partial sequences of endothelins, endothelin analogues or endothelin antagonists, which can be administered in vivo, preferably for the non-invasive imaging of vascular disorders.

Description

Our Ref: 494933 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT *o 00 0 0 9 Aftf 0*000* 0 0000 Applicant(s): Address for Service: Institut fur Diagnostikforschung GmbH an der Freien Universitat Berlin Spandauer Damm 130 D-14050 BERLIN

GERMANY

DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Invention Title: New agents for diagnosis of vascular diseases The following statement is a full description of this invention, including the best method of performing it known to me:- 5020

I

I

1 New Agents for the Diagnosis of Vascular Diseases This invention relates to the object characterized in the claims, i.e. metal complexes, metal complex conjugates of endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists, iodinated endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists, ani compositions that contain these comp-onds, their use as agents for diagnosis, and the methods for preparing these compounds and compositions. Metal complexes, metal complex conjugates of endothelines, endotheline derivatives, endotheline 15 partial sequences, endotheline analogues, or endotheline antagonists, as well as iodinated endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists that are applicable in vivo are used to yield images of vascular diseases, 20 preferably in a non-invasive way.

Atherosclerosis is a chronic, progressive disease of the blood vessels which has as yet been clinically S.diagnosable only at an advanced stage. Atherosclerotic vascular alterations are conventionally depicted by means of arteriography. A contrast medium is applied to the S relevant vessel using a catheter. X-rays then detect the vascular areas that show a constriction. It is one of this method's disadvantages that it is only suited for viewing subsections of the vascular system. As arteriography is an invasive method, its application may cause complications such as pains, perforations of arteries, arrhythmia, cardiac or cerebral infarcts which in unfavourite circumstances may result in the patient's death.

ID938EN.DOC Furthermore, methods based on the use of ultrasonic waves and MR tomography are used to diagnose atherosclerosis.

All methods currently applied have the great disadvantage that they detect atherosclerotic vascular changes from the diminished blood flow or significant alterations of the artery wall in these sections and are thus only capable of detecting advanced stages of atherogenesis.

Early detection of atherosclerosis would be of great importance for monitoring the therapeutic effect of diets, calcium antagonists, lipid and hypertension depressants, for monitoring restenosis after angioplastic surgery, for diagnosing coronary heart diseases, and for :15 detecting thrombotic deposits in the vessels.

Non-invasive methods of diagnosing atherosclerosis have been described before. Thus, antibodies labelled with radioisotopes, or labelled low-density lipoproteins :i"20 (LDL), were introduced that bond to atherosclerotic wall sections (Lees et al. 1983, J. Nucl. Med. 24, 154-156; Kaliman et al. 1985, Circulation, 72, 300; Virgolini et Sal. 1991, Eur. J. Nucl. Med., 18, 944-947). These methods, however, have major disadvantages such as the antigenic effect of the antibodies on the system and the long period of time (several days) required to isolate, refine, and label the LDL obtained from the patient's blood. Above all, these big molecules have a long halflif- in the blood, which together with high background radiation makes it difficult, if not impossible, to locate atherosclerotic lesions.

Shih et al. (1990, Proc. Natl. Acad. Sci., 87, 1436-1440) have synthesized partial sequences of the LDL protein moiety (apo-B-100) that still bond to the atherosclerotic plaques but have a considerably shorter half-life in the ID930KN DOC blood and an improved signal-noise ratio. A 'cessful in vivo diagnosis of atherosclerosis, however, .ould not be established with these apo-B-peptides due to their lower affinity to the plaque and/or lower density of the bonding places in the plaque.

It is, therefore, an object of the invention to provide new compounds and compositions that are suited for a new, non-invasive method of diagnosis of, in particular, the early, non-stenotic vascular diseases. This object is accomplished by the metal complexes and metal complex conjugates of endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists, as well as iodinated endotheline 15 derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists according to the invention.

Endothelines are physiologically active peptides that 20 have both hormonic and neuroregulatory functions within the organism (MacCumber et al. 1989, Proc. Natl. Acad.

Sci., 86,' 7285-7289; Yanagisawa et al. 1989, Trends Pharmacol. Sci., 10, 374-378; LeMonier de Gouville et al., 1989, Life Sci., 45, 1499-1513; Yanagisawa et al., 1988, Nature, 332, 411-415). Four different isotypes have as yet been found in man (Inoue et al., 1989, Proc. Natl.

Acad. Sci., 86, 2863-2867). Endotheline 1 is a polypeptide consisting of the following sequence of 21 amino acids: cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phecys-his-leu-asp-ile-ile-trp (Yanagisawa et al., 1988, Nature, 332, 411-415). An inactive precursor of endotheline, the big endotheline, is formed by the vascular endothelium. Endotheline, which ID93sEN.DOC bonds to specific receptors of the smooth vascular muscles, is formed after the endotheline-converting enzyme (ECE) has split off a heptadecapeptide. There, it results in a Ca++-mediated contraction of the smooth muscle cells (Yanagisawa et al., 1988, Nature, 332, 411- 415; Takuwa et al. 1991, Contrib. Nephrol., 90, 99-104).

The proliferation of smooth muscle cells in the vessel wall due among other to growth factors PDGF) is one of the early irreversible changes that occur during atherogenesis (Desmouliere and Gabbiani 1992, Cerebrovasc. Dis., 2, 63-71; Ross 1986, N. Engl. J. Med., 314, 448-501). These proliferating cells alter their morphology and their physiological function (Desmouliere and Gabbiani 1992, Cerebrovasc. Dis., 2, 63-71). The invitro incubation of atheromatous human coronary arteries with 12 SI-endothelin; 1 showed that increased bonding of 125I-endotheline 1 occurs in the tunica media and the vasa vasora sections (Dashwood et al., 1991, J.

20 Cardiovasc. Pharmacol., 17, 458-462). An increased uptake of 12 5 I-endotheline 1 in the deendotheliated aortic sections was found when 12 5 I-endotheline 1 was applied to rabbits whose abdominal aorta had been deendotheliated using a balloon catheter. This indicates a higher density of bonding places for endotheline 1 in these injured vascular sections (Kurata et al. 1992, J. Nucl. Med., 33, 845). The studies indicate that the proliferating smooth muscle cells keep expressing the endotheline receptor.

Endotheline 1 has a strong vasoconstrictive effect on the smooth vascular musculature M. Doherty, 1992, Medicinal Chemistry, 35, 1493-1508). Therefore, only relatively low concentrations may be applied i.v. to the organism. Higher concentrations may be applied of endotheline partial sequences, endotheline analogues or endotheline antagonists, as they still bond to the ID93BEN.DOC endotheline receptor but do not cause such a distinct contraction of the smooth muscle cells.

As endothelines are rapidly eliminated via the kidneys, the disturbing background radiation due to endotheline uptake in other organs or tissues is extremely low.

It was found that radiolabelled endothelines, endotheline derivatives, endotheline partial sequences as well as appropriate endotheline antagonists and endotheline analogues accumulate to a surprisingly great extent in atherosclerotic vascular lesions, thereby reaching a concentration sufficient for visualization by a scintillation camera or any other appropriate apparatus 15 used in nuclear medicine. Another surprising finding is that the substances used according to the invention reach this concentration in vivo so rapidly, and that the bond is so stable, that a concentration sufficient for diagnostics remains after the surplus of endotheline 20 derivatives, antagonists, or other substances bonding to the receptor have been carried away and eliminated. It was still another surprise that the accumulation occurs preferably at the sections of the arterial wall that are to be diagnosed and varied in different kinds although endotheline is active on all vascular regions.

Moreover, the substances used according to the invention are particularly appropriate as they do not additionally and non-specifically accumulate in other tissues or organs, contrary to many other classes of substances and substances tested. This is decisive for their suitability as agents for diagnosis.

The metal complexes, metal complex conjugates of endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline ID938EN.DOC antagonists, iodinated endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists according to the invention, and the solutions prepared from them, meet the requirements mentioned to a surprisingly great extent. They have both higher accumulation in pathological vascular areas and better contrast characteristics due to more favourable pharmacokinetics than the agents for diagnosis that have previously been described for detecting vascular diseases. The practical application of the new substances according to the invention is facilitated by their high chemical stability.

Complexes of compounds according to the invention of the 15 general formula (I) E-L-(K)b

(I)

containing metal ions vith the atomic numbers 21-32, 37-39, 42-51, and 57-83, are characterized, in that E is a residue derived from endothelines, 25 endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists, or a residue derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists carrying free thiol groups and directly binding a metal ion; L is a direct linkage or a ZI-R-Zz residue, wherein R is a unbranched, branched, saturated or unsaturated C1- 2 0 -alkyl group, optionally ID93 8K.DOC interrupted by one or several oxygen and/or sulphur atoms and/or carbonyl, -NHCO-, -N(Ci.

6 alkyl)CO-, -NH- and alkyl) groups, and optionally substituted with hydroxy and/or epoxy groups;

Z

1 and Z 2 are, independent of each other, an or group, or a residue of formula a (CH2)t- Z2 -Z -(CH2)s B- (a) 15 wherein s and t are, independent of each other, the integers 0, 1, 2, or 3, ring B represents a phenyl or cyclohexyl group, and Z, and Z 2 have the abovementioned meaning, b represents the integers 0 or 1, SK is a chelating agent residue of the general formula II A or II B So R-0 0

R

2 S C C IA ID938HN.DOC 8 0 11 R3- S

(CH

2 )n 1 C NH

R

R

5 S (CH 2 C NH 11 0 I1 B) wherein

R

2

R

3 and R 5 are, independent of each other, a hydrogen atom, a (Cl- 6 alkyl)CO, (C 6 8 aryl)CO, or (C 7 9 arylalkyl)CO residue, optionally substituted with a hydroxyl group, a C 1 4 alkoxy, a carboxyl, or a suiphonic acid group, and

R

4 represents a residue of either formula II C or II D

CH

2 CH c=0 (lII C) 555 CH -(CH 2 2

(IID)

C-0 wherein the carbon atoms labelled with an asterisk are bound to the imino groups of formula II B, and wherein n' is an integer 1 or 2, ID93SEN.DOC i is any integer from 2 to 6, and TT represents a- and/or P-amino acids linked together in the usual way through amide bonds; as well as chelating agent residues derived from dithiosemicarbazone derivatives of formula II E Rp HN-N N-NH R (I I E) N= N 1io SH HS wherein

R

6 is a hydrogen atom or a C1-6 alkyl group; as well as chelating agent residues derived from bis(aminothiol) derivatives of formula II F

RR

P

R R NH NH

RF)

R9 -RI (I I F) R7 SH HS R8 wherein

R

7 to R 18 represent, independent of each other, a hydrogen atom, a C1-lo alkyl chain and/or an Llinkage, while o, p, r are either integers 1 or 2; ID93BEN,DOC as well as chelating agent residues derived from propylene aminoxime derivatives of formula II G

R

21 NH NH R22 R 2 0 "R 23

I

R,9 'N

OH

1 .o oooo o**o °o01 o oo wherein

RI

9 to R 24 represent a hydrogen atom or a CI.

4 alkyl residue, either identical or different, and independent of each other, m' is either integer 2 or 3; as well as chelating agent residues derived from diamido-dimercapto derivatives of formula II H

S

a

S.

Y

1 Y2 NH X, HN, A S

A

2 S S I I IIH wherein X, is a bond, a methylene group, or a CHY 4 group, with one of groups Y 1

Y

2

Y

3 or Y4 represei.:ing an L-linkage and the others hydrogen atoms or optionally an oxygen atom, ID938EN.DOC T is a hydrogen atom, an alkaline metal ion, a

C.-

6 acyl group, a benzoyl group, a hydroxyacetyl group, an acetamido methyl group, ,a p-methoxy benzyl group, an ethoxy ethyl group, or any other suitable SH protective group,

A

2

A

3 and A 4 are hydrogen atoms or C_-6 alkyl groups, either identical or different, and independent of each other other; as well as chelating agent residues derived from diamido-dimercapto derivatives of formula II J R27 S. R 2 5

R

2

.R

26

R

26

ZZ

wherein R27 represents a hydrogen atom, or a Cz 6 alkyl residue optionally substituted with one or two hydroxyl groups, N.H: 20N wherein

R

2 7 represents a hydrogen atom, or a C 1 -6 alkyl residue optionally substituted with one or two hydroxyl groups,

R

25 and R 26 are a hydrogen atom each, or an oxygen atom together A is a hydroxyl or mercapto group, Y is a hydrogen atom, a carboxy or sulphonyl residue, and Z is a carbon atom or a nitrogen atom; ID938EN.DOC as well as chelating agent residues derived from amino polycarboxylic acids of either formula II K or II L

CH

2 CO- CH 2 X CH 2 X CH 2

X

I I I I N (CH 2

-CH

2

CH

2

CH

2 (N-CH2-CH 2 m-N I K)

CH

2 X CH 2

X

U R,

CH

2

X

N (CH 2 CH (CH 2

N

*I O

:I

*CH I I N ((CH 2 0

CH

2 X

CH

2

X

10 wherein n and m are either integer 0, 1, 2, 3, or 4, with n plus m not exceeding 4, oa is an integer 2, 3, 4, or k is an integer 1, 2, 3, 4, or 1 is an integer 0, 1, 2, 3, 4, or 5, and q is an integer 0, 1, or 2, e"e" U represents a hydrogen atom, or a C.- 6 alkyl group optionally substituted with one or several hydroxy groups and containing an L-linkage, each residue X represents hydrogen atoms, COOH groups, ester groups, or amide groups with 1 to 6 carbon atoms in the alkyl residue, independent of each other, RI is an L-linkage or a hydrogen atom; ID938RN.DOC 13 as well as chelating agent residues of formula II

M,

Cp(aa)Cp- (II M) wherein Cp is a protected cysteine and (aa) one of the naturally occurring amino acids; as well as cysteine-rich amino acid sequences of the metalloid thionines -ser-cys-thr-cys-thr-ser-ser-cys-ala-, o -ala-cys-lys-ala-cys-lys-cys-, -gly-cys-ser-lys-cys-ala-gln-gly-cys-val-, 15 -cys-lys-gly-ala-ala-asp-lys-cys-thr-cys-cysalaand analogous sequences in which serine has been replaced by threonine, glycine, or alanine; as well as endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists that are labelled using radioactive iodine isotopes.

The metal ions contained in the complexes according to the invention have properties that facilitate their identification using physical methods. These said properties are, for example, paramagnetism, radioactivity including and y-radiation, and a large absorption cross section for X-rays and other radiation. This is true, in particular, of the transitional elements, the lanthanoids, and the main group elements of the 4th to 6th periods.

ID938EN.DOC Ions of isotopes of the following metals are particularly preferred due to the said properties: Tc, Re, In, Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sa, Y, Gd, Tb, Dy, Ho, Er, and La. Moreover, some iodine isotopes have the said properties.

The complexes according to the invention of compounds of the general formula (I) .0 E-L- (K)b (I) :15 'r :V 20 containing metal ions with the atomic numbers 21-32, 37- 39, 42-51, and 57-83, further contain residues E :hich derive from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists. These residues E have the property to bond selectively to endotheline receptors.

The residues E preferably derive from endothelines.

Endotheline 1 is particularly preferred among the endothelines. It comprises the following sequence of amino acids (or parts thereof): cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phecys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe- *0 *r cys-his-leu-asp-ile-ile-trp.

The hydrophobic region of endotheline 1, his16-trp 2 1 is essential for bonding to the receptor (Kimura et al., 1988, Biochem. Biophys. Res. Comm., 156, 1182-1186).

Furthermore, residues are preferred that derive from endotheline partial sequences.

ID938EN.DOC Therefore, peptides that include the -his-leu-asp-ileile-trp-sequence of amino acids are particularly preferred among the endotheline partial sequences used according to the invention.

Furthermore, residues E are preferred that derive from endothe line analogues.

The particularly preferred endotheline analogues according to the invention comprise the following sequences of amino acids or parts thereof: *e S *0*0

S

S

5 S. 55 o S

S.

S S -cys-ser-cys- ser-ser-trp-leu-asp-lys-glu-cys-val -tyr-phecys-his-leu-asp-ile-ile-trp- (endotheline 2) -cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu--cys-val-tyr-tyr- 20 1 cys-his-leu-asp-ile-ile-trp- (endotheline 3) -cys-ser-ala-ser- ser-leu-met-asp-lys--glu-ala-val -tyr-phe- 25 cys-1.;i -leu-asp-ile-ile-trp- (ala-endotheline) 5S SS S *5 S S S

SS

S

0S@S~S

S

-cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr-phecys-his-leu-asp-ile-ile-trp- (VIC) -cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn-phecys-his-gln-asp-val-ile-trp- (sarafotoxin S 6a,) 10933514.DOC I I -ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-pheala-his-leu-asp-ile-ile-trp- -ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-pheala-his-leu-asp-ile-ile-trp- -cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val-tyr-pheala-his-leu-asp-ile-ile-trp- Furthermore, residues E are preferred that derive from endotheline antagonists.

S 15 Preferably used endotheline antagonists are cyclic pentapeptides such as: -D trp-D asp-pro-D val-leu- or -D glu-ala-allo D ile-leu-D trp-.

Furthermore, the complexes according to the invention of compounds of the general formula (I) E-L-(K)b (I) containing metal ions with the atomic numbers 21-32, 37- 39, 42-51, and 57-83, contain residues L that link the complexing agent residues K, which themselves bind the respective metal ions, with the residues E derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists.

In preferred complexes according to the invention, residue L is an unbranched, branched, cyclic, aliphatic, ID938BE.DOC aromatic, or arylaliphatic alkylene residue with up to carbon atoms.

Furthermore, complexes according to the invention are preferred in which L stands for ZI-R-Z 2 wherein are:

Z

1 and Z 2 independent of each other, a group, and R an unbranched mono- to decamethylene group or for a residue of formula c I

(CH^-Z,

Z, -(CH 2 wherein equals 1 and t equals 0, ring B is phenylene, and Z, and Z 2 represent, independent of each other, a 20 -NH-, group.

The complexing agent residues K that are also contained in the complexes according to the invention must be able to bind the respective metal ions through coordinate or covalent bonds.

Depending on the type of metal ion to be bound, on its charge, oxidation number, and atom or ion radius, the complexing agent residue K should be selected to create an effective bond sufficient for putting the complexes according to the invention to the use according to the invention.

iD938MaDOC Preferred complexing agent residues K are: 4-carboxyethylphenylglyoxal-bis- (N-methylthio-semicarbazone) -N-hydroxysuccinimide ester, 6- -isothiocyanatobenzyl) -3,3,9,9-tetramethyl-4,8-diaza-undecane-2,10dion-dioxime, 2-methyl-2- (4-isothiocyanatobenzyl) propylene-bis-salicylidenamine, 2-methyl-2- (4-isothiocyanatobenzyl) -propylene-bis- (suipho) salicylidenamine, N,N' -bis [2-mercaptopyridyl) methyl] -2methyl-2- (4-isothiocyanatobenzyl) 3-propanediamine, Sbenzoylthioacetylglycylglycyl glycine, N, N'-bis (benzoylthioacetyl) 3-diaminopropionic acid, N,N' -bis (benzoylthioacetyl) 4-diaminobutyric acid, N,N' -bis (benzoylthioacetyl) -4,5-diaminopentanoic acid, N,N' -1,2-ethylenediyl-bis- (2-mercapto-l-carboxy-ethylamine), Cys (Acm) Gly- Cys (Acm) GlyGlyArgGlyAspSer, ethylenediaminotetraacetic acid, diethylenetriaminopentaacetic acid, trans-1,2cyclohexane diaminotetraacetic acid, l,4,7,10-tetraazacyclododecanetetraacetic acid, 1,4, 7-triazacyclononanetriacetic acid, 1,4,8, 1l-tetraazatetradecane tetraacetic acid, 1,5,9-triazacyclododecanetriacetic acid, 1,4,7,10tetraazacyclododecanetriacetic ac'~d, and 3,6, 9,15-tetraazabicyclo[9,3, 15] -pentadeca-1 13-trien-triacetic acid. If desired, a part of the carboxylic acids may be present as asters and/or amides, If at least a part of the carboxylic acid groups is to be in the form of amides, tertiary amides are preferred. The residues may be saturated, unsaturated, unbranched or branched-chain, or cyclic hydrocarbons with up to 5 C atoms which may be optionally substituted, with 1 to 3 *hydroxy or C 1

-C

4 alkoxy groups. The following groups shall be named as examples: methyl, ethyl, 2-hydroxy- *ethyl, 2-hydroxy-1- (hydroxymethyl) -ethyl, 1- (hydroxymetlthl, propyl, isopropenyl, 2 -hydroxypropyl, 3hydroxypropyl, 2,3-dihydroxypropyl, butyl, isobutyl, isobutenyl, 2-hydroxybutyl, 3-hydroxybutyl, and XD938amq.rOC 4-hydroxy-2-methylbutyl, 2- and 3-hydroxyisobutyl, 2,3,4trihydroxybutyl, 1,2, 4-trihydroxybutyl, pentyl, cyclopentyl, and 2-methoxyethy. group. The amide residue may also be a heterocyclic 5- or 6-membered ring formed with an enclosure of the amide nitrogen. The following rings shall be named as examples: the pyrrolidinyl, piperidinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, morpholinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl ring.

Particularly preferred embodiments of complexes of compounds of the general formula (I) E-L-(K)b (I) containing metal ions with the atomic numbers 21-32, 37- 39, 42-51, and 57-83, are, for example, 2 a) 9 9 mTc complex of S -benzoylthioacetyl -gly-gly-gly-gly-asp-his- leu-aspile-ile-trp, b) 99 mTc complex of :25 N,N' -bis (S-benzoylthioacetyl) -3,4-diaminobutyrylcys -ser-cys-ser-ser-leu-met -asp-lys-glu-cys-val-tyrphe-cys-his-leu-asp-ile-ile-t rp, c) 99 mTc complex of 3- [p-phenylglyoxal-di (N-methylthio semicarbazone)] I I propionyl-cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-

H--

val-tyr-phe-cys-his-leu-asp-ile-ile-trp, ZD93SM4.*DOC d) 99 mTc complex of 3,6-diaza-1,8-dimercapto-2,7-bis- (carbonyl-gly--his-leu-asp-ile-ile-trp) octane, e) 99 mTc complex of ,3,9,9-tetramethyl-4,8-diazaundecyl-2, 1O-dion-dioxime) -6-yljmethylphen-4' -yl} arninothiocarbonyl -cys--ser-ala-ser-ser-leu-met -asp-lys f) 99mTc complex of N,N' -bis (S-benzoylthioa ,,i-tyl) -4,5-diamino-1-oxopentyl -cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cysval-t~rr-phe-cys-his-leu-asp-ile-ile-trp, g) 99 mTc complex of {[2,6-diaza-1,7-di(2-hydroxyphenyl) -4methyl-hept-4-yl]-methyl-phen-4' -yl} aminothio carbonyl-cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cysval-tyr-tyr.-cys-his-leu-asp-ile-ile-trp, 2 h) 9 9 mTc complex of {[2,6-diaza-1,7-di(2-hydroxy-5suiphophenyl) -4-methyl-hept-4-yl]-methylphen-4' -yl} aminothiocarbonyl-ala-ser-ala-ser-ser-leu-met-asp-lysglu-ala-vai -tyr-phe-ala-his-leu-asp- ile-ile-trp, j) 9 9 mTc complex of N-[3,6,9-triaza-l-oxo-3,6,9,9-tetra- (hydroxycarbonylmethyl) -nonyl]-gly-his-leu-asp-ileii e -trp, k) 11 1 In complex of 6, 9-triaza-l-oxo-3, 6, 9, 9-tetra- (hydroxycarbonylmethyl) -nonyl]-gly-his-leu-asp-ilei le -trp, ID93SEN.DOC 1) Gd(III) complex of N-[3,6,9-triaza-1-oxo-3,6,9,9tetra- (hydroxycarbonylmethyl) -nonylj-gly-his leu- aspile-ile-trp, sodium salt, m) Gd(III) complex of 1-{2-hydroxy-3-[4-(gly-his-leu-aspile-ile-trp-thiouridyl)phenoxylpropyl} 1,4,7,10tetraaza-4, 7, 10-tris- (carboxylatomethyl) -cyclododecane, n) 99mr~c complex of. cys (acm) -gly-cys (acm) cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyro) 9 9 mTc complex of of cyclo(trp-leu-val-pro-asp)cys (acm) -gly-cys (acm), 20 p) 99 mTc complex of 4-mercaptobutyrimidyl-cys-ser-alaser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-hisleu-asp-ile-ile-trp, r) 99 mTc complex of 3-thiopropionyl-gly-asp-his-leu-aspile-ile-trp, s) 99 m'rc complex of 2-(acetylthio)succinyl-gly-asp-hisleu-asp-ile-ile-trp, t) 99 mTc complex of cys-ser-cys-ser-ser-leu-met-asp-lysglu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp.

Among the particularly preferred complexes according to the invention of compounds of the general formula (I) M9 3m E. DC containing metal ions, are further such complexes in which b takes the value 0 and L represents a bond.

The metal ions are bound to free thiol groups that are present in the residues E derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists.

For example, preferred metal ions are 99 mTc and 186 Re.

Other preferred compounds according to the invention are endotheline derivatives, endotheline partial sequences, S **o endotheline analogues or endotheline antagonists labelled with radioactive iodine isotopes. The said iodine isotopes have radioactive properties, which makes them detectable by physical methods. Particularly preferred iodine isotopes are 131I, 125I, or 123I.

Another object of the present invention are new 20 conjugates between the complexing agent residue K and residue E, derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists that are linked with one another through residue L and formed according to the 25 general formula (I) E-L-(K)b (I) wherein: E is a residue derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists,; L is a direkt linkage or a ZI-R-Z 2 residue, wherein ID938EN.DOC R is a unbranched, branched, saturated or unsaturated C.1 2 0 -alkyl group, optionally interrupted by one or several oxygen and/or sulphur atoms and/or carbonyl, -NHCO-, -N(Ci-6 alkyl)CO-, -NH- and -N(Cl.

6 alkyl) groups, and optionally substituted with hydroxy and/or epoxy groups; Z, and Z 2 are, independent of each other, an or group, or a residue of formula a (CHA) Z2 Z, B- (a) s and t are, independent of each other, the intege A or II 2, or 3,B 0 0 b represents the integers 0 or 1, K is a chelating agent residue of the general formula II A or II B 0 0

R

2 S C C I A) ID938E1.DOC 0 11

R

3 S

(CH

2 C NH

R

0 IIB wherein

R

2

R

3 and RS are, independent of each other, a hydrogen atom, a (C 1 6 alkyl)CO, (C 6 8 aryl)CO, or

(C

7 arylalkyl)CO residue, optionally substituted with a hydroxyl group, a C 1 4 alkoxy, a carboxyl, or a suiphonic acid group, and

R

4 represents a residue of either formula II C or II D

CH

2 -CH

I~

I 0) a a a *.aaaa a -CH (cH 2 2

C=O

II D) wherein *the carbon atoms labelled with an asterisk(* are bound to the imino groups of formula II B, and wherein n' is an integer 1 or 2, i is any integer from 2 to 6, and WD39JSf.WC TT represents c- and/or P-amino acids linked together in the usual way through amide bonds; as well as chelating agent residues derived from dithio semicarbazone derivatives of formula II E /4 R HN-N N-NH R (I E

==N

SH HS S10 wherein 0 0. R 6 is a hydrogen atom or a C1-6 alkyl group; 0 as well as chelating agent residues derived from bis(aminothiol) derivatives of formula II F R, R4 R NH NH R1 .0 R R"R, (I IF) R e R 1

R

7 SH HS R wherein

R

7 to R 18 represent, independent of each other, a hydrogen atom, a C1-lo alkyl chain and/or an Llinkage, while o, p, r are either integers 1 or 2; as well as chelating agent residues derived from propylene aminoxime derivatives of formula II G ID93BBH.DOC

S(

I

G)

R

1 9 N N R 24 I I OH OH wherein

R

19 to R 24 represent a hydrogen atom or a CI.

4 alkyl residue, either identical or different, and independent of each other, m' is either integer 2 or 3;

S

e e* as well as chelating agent residues derived from 10 diamido-dimercapto derivatives of formula II H

Y

1 Y2 NH X HN Y3 I H A, A 3

A

2 S S A 4 T T wherein

X

1 is a bond, a methylene group, or a CHY 4 group, with one of groups YI, Y 2

Y

3 or Y 4 representing an L-linkage and the others hydrogen atoms or an oxygen atom, if any, T is a hydrogen atom, an alkaline metal ion, a CI.

6 acyl group, a benzoyl group, a hydroxyacetyl group, an acetamido methyl group, a p-methoxy benzyl ID938 i.DOC group, an ethoxy ethyl group, or any other suitable SH protective group,

A

2

A

3 and A 4 are hydrogen atoms or Cl-6 alkyl groups, either identical or different, and independent of each other; as well as chelating agent residues derived from diamido-dimercapto derivatives of formula II J

R

25

R

2 Ric RR 2 6 NH NH 26 N I I J A A wherein

SR

27 represents a hydrcgen atom, or a C.16 alkyl residue optionally substituted with one or two hydroxyl groups,

R

25 and R 26 are a hydrogen atom each, or an oxygen S atom together A is a hydroxyl or mercapto group, Y is a hydrogen atom, a carboxy or sulphonyl residue, and Z is a carbon atom or a nitrogen atom; as well as chelating agent residues derived from amino polycarboxylic acids of either formula II K or II L ID9381N.DOC

CH

2 CQ- CH 2 X HXC2 N -(CH 2

-CH

2

CH

2 Cl 2

(N-CH

2 -CHA) N

CH

2 X

CH

2

X

(II K U R I I I N (CH 2 CHI (CH 2 1

N

I CHO

(('CH

2 6 12X

~H

2 o -Nq

CH

2

X

I IL 1 5 200

CH

2

X

wherein n and m are either integer 0, 1, 2, 3, or 4, with n plus m not exceeding 4, a is an integer 2, 3, 4, or k is an integer 1, 2, 3, 4, or 1l. s an integer 0, 1, 2, 3, 4, or 5, and q is an integer 0, 1, or 2, U represents a hydrogen atom, or a CI- 6 alkyl group optionally substituted with one or seve~ral hydroxy groups and containing an L-linkage, each residue X represents hydrogen atoms, COON groups, ester groups, or amide groups witb 1 to 6 carbon atoms in the alkyl residue, independent of~ each other, R, is an L-linkage or a hydrogen atom; as well as chelating agent residues of formula II M, Cp (aa) Cp- (I I M) wherein WD9)1D1.WC Cp is a protected cysteine and (aa) one of the naturally occurring amino acids; as well as cysteine-rich amino acid sequences of the metalloid thionines -ser-cys-thr-cys-thr-ser-ser-cys-a'la-, -ala-cys-lys-ala-cys-lys-cys-, -gly-cys-ser-lys-cys-ala-gln-gly-cys-val-, .ys-lys-gly-ala-ala-asp-lys-cys-thr-cys-cys-alaand analogous sequencCG in which serine has been replaced by threonine, glycine, or alanine.

*foe The residues E of the complexes according to the 15 invention of compounds of the general formula (I) (K)b (I) preferably derive from endothelines.

to Endotheline 1 is particularly preferred among the endothelines. It comprises the follow'ing sequence of amino acids (or parts thereof): cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phiecys-his-leu-asp-ile-ile-trp.

The hydrophobic region of endotheline 1, his1 6 -trp 2 1 is essential for bonding to the receptor (Kimura et al., 1988, Biochem. Biophys. Res. Comm., 156, 1182-1186).

Furthermore, residues are preferred that derive from endotheline partial sequences.

WD93OM.DOC Therefore, peptides that include the -his-2.eu-asp-ileile -trp- sequence of amino acids are particularly preferred among the endotheline partial sequences used according to the invention.

Furthermore, residues E are preferred that derive from endotheline analomjvza.

The particularly preferred endotheline analogues according to the invention comprise the following sequences of amino acids or parts thereof: 0 04 6406 sees :2 5 I I cys -se r -cys ser ser -trp 1eu -asp -lys -glu -cys -va1- tyr-p he cys-his-leu-asp-ile-ile-trp- (endotheline 2) I I -cys- thr-cys -phe-thr-tyr-lys-asp-lys-glu-cys-val -tyr-tyrcys -his- leu-asp- ile-ile- trp- (endotheline 3) -cys -ser-ala-ser-ser-leu-met -asp-lys-glu-ala-val -tyr-phecys -his -leu-asp-ile -ile-trp- (ala-endotheline>, -cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr-phecys-his-leu-asp-ile-ile-trp- (VIC) -cys -ser-cys-lys -asp-met-thr-asp-lys-glu-cys-leu-asn-phecys -his -gln-asp-val- ile- trp- (sarafotoxin S 6a) ID93SE.OC -ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-pheala-his-leu-asp-ile-ile-trp- -ala-ser-ala-ser-ser--leu-met-asp-lys-glu-ala-val-tyr-pheala-his-leu-asp-ile-ile-trp- -cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val -tyr-poheala-his-leu-asp-ile-ile-trp- Furthermore, residues E are preferred that derive from .:015 Preferably used endotheline antagonists are cyclic pentapeptides such as: -D trp-D asp-pro-D val-leu- or -D glu-ala-allo D ile-leu-D trp-.

The complexes according to the invention of compounds of the general formula (I) 2 E-L-(K)b (I) preferably contain residues L that are unbranched, branched, cyclic, aliphatic, aromatic, or arylaliphatic and have up to 20 carbon atoms.

Furthermore, preferred residues L stand for Z 1

-R-Z

2 wherein are: Z, and Z 2 independent of each other, a group, and R is an unbranched mono- to decamethylene group or for a residue of formula cL

(CH

2 Z 2

Z

1

-(H

25 s wherein s equals 1 and t equals 0, and ring B is phenylene and wherein are and Z 2 independent of each other, a -NH- -NH- NH-, -NH- group.

Preferred complexing agent residues K of compounds to 0 according to the invention of the general formula (I) derive from 4-carboxyethylphenylglyoxal-bis- (N-methylthio-semicarbazone) -N-hydroxysuccinimide ester, 6- isothiocyanatobenzyl) 9-tetramethyl-4, 8-diazaundecane-2, lo-dion-dioxime, 2-methyl-2- (4-isothiocyanatobenzyl) -propylene-bis-salicylidenamine, 2-methyl- 2- (4-isothiocyanatobenzyl) -propylene-bis- (suipho) salicylidenamine, N,N' -bis [2-mercaptopyridyl) methyl] -2-methyl-2- (4-isothiocyanatobenzyl) -1,3propanediamine, S-benzoylthioacetylglycylglycyl glycine, NI'-bis (benzoylthioacetyl) 3-diaminopropionic acid, N,N' -bis (benzoylthioacetyl) 4-diaminobutyric acid, N,N' -bis (benzoylthioacetyl) -4,5-diaminopentanoic acid, N,N' -1,2Lethylene-diyl-bis- (2-mercapto-i-carboxyethylamine), Cys (Acm) GlyCys (Acm) GlyGlyArgGlyAspSer, ethylenediaminotetraacetic acid, diethylenetriaminopentaacetic acid, trans-i, 2-cyclohexane diaminotetraacetic acid, 1,4,7, acid, 1,4,7-triazacyclononanetriacetic acid, 1,4,8,11- ID3)$WidJOC tetraazatetradecane tetraacetic acid, 1,5,9-triazacyclododecanetriacetic acid, 1,4,7,10-tetraazacyclododecanetriacetic acid, and 3,6,9,15-tetraazabicyclo[9,3,15]pentadeca-1(15),11,13-trien-triacetic acid. If desired, a part of the carboxylic acids may be present as esters and/or amides.

If at least a part of the carboxylic acid groups is to be in the form of amides, tertiary amides are preferred. The residues may be saturated, unsaturated, unbranched or branched-chain, or cyclic hydrocarbons with up to 5 C atoms which may be optionally substituted, with 1 to 3 hydroxy or C 1

-C

4 alkoxy groups. The following groups shall be named as examples: methyl, ethyl, 2-hydroxy- 15 ethyl, 2-hydroxy-l-(hydroxymethyl) -ethyl, 1-(hydroxymethyl)-ethyl, propyl, isopropenyl, 2-hydroxypropyl, 3hydroxypropyl, 2,3-dihydroxypropyl, butyl, isobutyl, isobutenyl, 2-hydroxybutyl, 3-hydroxybutyl, and 4-hydroxy-2-methylbutyl, 2- and 3-hydroxyisobutyl, 2,3,4- 20 trihydroxybutyl, 1,2,4-trihydroxybutyl, pentyl, cyclopentyl, and 2-methoxyethyl group. The amide residue may also be a heterocyclic 5- or 6-membered ring formed with an enclosure of the amide nitrogen. The following rings shall be named as examples: the pyrrolidinyl, 25 piperidinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, morpholinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl ring.

The complexes of compounds according to the invention of the general formula containing metal ions are prepared using a method known in the art, in which a radioactive metal ion in the form of its permetallate reacts with a compound of the general formula (I) E-L- (K)b (1) I'S3 I8 .DOC in the known way, with a reducing agent and optionally an auxiliary ligand, being present.

Preferred metal ions are 99 mTc or Re in the form of pertechnate or perrhenate.

The reaction is preferably carried out in a hydrous medium at room temperature. The SH protective groups are cleaved in situ or according to the methods known to a person skilled in the art from literature, for example, using basic hydrolysis, reductive decomposition, etc.

(see, for example, "Protective Groups in Organic Synthesis", T. W. Greene, John Wiley and Sons 1981).

The complexes of compounds according to the invention of the general formula containing metal ions are further prepared by reacting a suitable salt or oxide of a suitable paramagnetic or radioactive cation in a way known to a person skilled in the art with a compound of the general formula (I) E-L- (K)b Among the preferred radioactive metal ions is, for example 11 1In, preferred metal ions having paramagnetic properties are the ions of Gd.

The paramagnetic cations are incorporated in the chelating agent residues II K and II L as known from literature (see, for example, DE 34 01 052 and EP 430863),' where the metal oxide or salt nitrate, acetate, carbonate, chloride, or sulphate) of the desired metal is suspended or dissolved in polar solvents such as water or hydrous alcohols, and reacted with the appropriate amount of complexing ligands. If desired, existing acidic hydrogen atoms or acid groups may be ID9381.DOC substituted by cations of inorganic and/or organic bases or amino acids.

Among the appropriate inorganic cations are, for example, the lithium ion, the potassium ion, the calcium ion and, in particular, the sodium ion. Appropriate cations of organic bases are, among others, ions of primary, secondary, or tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethyl glucamine and, in particular, N-methyl glucamine. Among the appropriate cations of amino acids are, for example, those of lysine, arginine, and ornithine, as well as amides of otherwise acidic or neutral amino acids.

.oeeo 15 The complexes according to the invention of compounds of the general formula containing metal ions are further prepared by cleaving the disulphide bridges that are present in the endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists in the way known in the art, or by generating or introducing free thiol groups that react with the radioactive metal ion in the form of a permetallate in the presence of a reducing agent and optionally an auxiliary ligand using suitable methods '.i25 such as the Trauts method.

e Metal complexes of compounds of the general formula (I) on the condition that L represents a bound and b takes the value 0, are prepared in the ways known from literature, for example, by reducing the disulphide bridges present in E' that stands for endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists with 2-mercaptoethanol according to the method described in EP 0 271 806 or in Nucl.Med.Biol. 12, 607-609, 1992, and by labelling them using technetium-99m in the presence of auxiliary ID938I NOC ligands such as pyrophosphate, gluconate, or glucoheptonate and a reducing agent in a way known in the art, or by reacting E' with suitable heterobifunctional crosslinkers as, for example, N-succinimidyl-3-(2-pyridyldithio) propionate, that are capable of forming an amide linkage with a terminal amino group or, optionally, an amino group of a properly substituted side-chain in E', and that have, on the other hand, a sulphur group, optionally protected; then splitting off the protective sulphur group in a way known in the art and labelling it in a way known in the art using technetium-99m or Re, or by reacting E' with suitable derivational reagents known from literature, such as imino thiolane, and by labelling it then with technetium-99m or Re in a way known in the 15 art.

The iodinated endotheline derivatives, andotheline partial sequences, endotheline analogues or endotheline antagonists can be prepared preferably with 131I, 1251, or 20 123I using methods such as the chloramine-T method (Krohn et al., 1977, BBA 490, 49), the lactoperoxidase method (Kitamura et al., 1990, Biochem. Biophys. Res. Commun.

497-503; Emori et al., 1990, Biochem. Biophys. Res.

Commun. 162, 217-223), the Bolton-Hunter method (Bolton 25 and Hunter, 1973, Biochem. 133, 529-539), the iodogen .method (Fraker and Speck, 1978, Biophys. Res. Commun., 849-857), or other known methods.

The compounds according to the invention of the general formula are prepared in a way known in the art by reacting a functional group in E' with a functional group in the general formula (III) (B)b-L-H (III) wherein ID938EN.DOC L, K, and b have the meanings specified above.

The compounds of formula are prepared using a method known to a person skilled in the art by reacting a functional group in L, for example, with a terminal or amino group, or a side-chain correspondingly substituted in K' is linked to E' covalently, preferably through an amine or thioamine linkage, where E' represents endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists, and K' represents chelating agents of the general formulae II A to II M.

S If the chelating agents K' are linked through a carboxyl 15 group in L, the latter is transformed according to the methods known to a person skilled in the art, for example, the carbodiimide method (Fieser, Reagents for Organic Synthesis 10, 142), or by means of a mixed or cyclic anhydride (Org. Prep. Proc. Int. 1975, 2, 215), or an activated ester (Adv. Org. Chem. Part B, 472) and successive reaction with a nucleophile group in E', preferably an amino group, with the formation of a covalent bond.

Isothiocyanate and a-halogenacetyl derived complexing agents or complexes under pH control are also reacted in ways known from literature in a hydrous environment with the desired amine-containing E' representing endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists.

E' is prepared according to the methods described by Barany and Marrifield in The Peptides: Analysis, Synthesis, Biology. Academic Press, New York, 1980; ID938 DOC Stewart and Young, Solid-phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL, 1984.

Chelating agents K' of the general formula II A are prepared as known from literature (see EP 0248506) by chloracetylating N-terminal amino groups of di-, tri-, tetra-, penta-, or hexapeptides and successive reaction of the resulting N-chloracetyl peptides with alkali salts of thiocarbonic acids. Another method for preparing chelating agents K' of the general formula II A as known from literature (see EP 0248506) consists in reacting suitable activated (for example, NHS ester) and Se* acylated derivatives of thioacidic acid or 3thiopropionic acid with di-, tri-, tetra-, penta-, or hexapeptides. The respective carbonic acids are activated Sby a method known to a person skilled in the art, for S example, according to the carbodiimide method (Fieser, Reagents for Organic Synthesis 10, 142), or using a mixed or cyclic anhydride (Org. Prep. Proc. Int. 1975, 2, 215) 20 or an activated ester (Adv. Org. Chem. Part B, 472).

S Chelating agents K' of the general formula II B are prepared as known from literature (see EP 0248506) by chloroacetylating the free amino groups of 1,2- 25 diaminopropionic acid or 1,3-diaminobutyric acid and 0* successive reaction of the resulting N,N'-dichloroacetyl diaminocarbonic acids with alkali salts of thiocarbonic acids. Another method for preparing chelating agents K' of the general formula II B as known from literature (see EP 0248506) consists in reacting suitable activated (for example, NHS ester) and S-acylated derivatives of thioacidic acid or 3-thiopropionic acid with 1,2diaminopropionic acid or 1,3-diaminobutyric acid.

The respective carbonic acids are activated by a method known to a person skilled in the art, for example, ID938KNDOC according to the carbodiimide method (Fieser, Reagents for Organic Synthesis 10, 142), or using a mixed or cyclic anhydride (Org. Prep. Proc. Int. 1975, 2, 215) or an activated ester (Adv. Org. Chem. Part B, 472).

Chelating agents X' of the general formula II E are prepared as known from literature by reacting a substituted 1,2-dicarbonyl compound with a thiosemicarbacide as described in European Patent Application EP 0 306 168.

Chelating agents K' of the general formula II F are S. principally prepared by reductive aminations of substituted or non-substituted 1,2-diketo or 1,3-diketo compounds with substituted, unsubstituted, protected or S. unprotected aminothiols as described in EP 279 417.

Chelating agents K' of the general formula II G are prepared as known from literature by reacting 2- 20 substituted 1,3-propane diamines with 2-chlorine-2-alkyl- 3-nitrosoalkanes as described in the European Patent Applications EP 0417 870 and EP 0 502 594, or by reacting 2-substituted 1,3-propane diamines with the respective imines that are reduced to the respective amines in the 25 way known in the art.

S

Chelating agents K' of the general formula II H are prepared as known from literature (see US 4,897,255) by chloracetylating the free amino groups of 1,2- or 1,3diaminoalkanoic acid (see EP 0248506) and successive reaction of the resulting N,N'-dichloroacetyl diaminocarbonic acids with alkali salts of thiocarbonic acids. Another method for preparing chelating agents K' of the general formula II H as known fror literature (see US 4,897,255) consists in reacting suitable activated (for example, NHS ester) and S-acylated derivatives of ID938EN.DOC thioacidic acid or 3-thiopropionic acid with diaminopropionic acid or 1,3-diaminobutyric acid. The respective carbonic acids are activated by a method known to a person skilled in the art, for example, according to the carbodiimide method (Fieser, Reagents for Organic Synthesis 10, 142), or using a mixed or cyclic anhydride (Org. Prep. Proc. Int. 1975, 2, 215) or an activated ester (Adv. O-g. Chem. Part B, 472). The separation of the SH protective groups is done after coupling to E' according to the methods known to a person skilled in the art from literature, for example, using basic hydrolysis, reductive decomposition, etc. (see, for example, "Protective Groups in Organic Synthesis", T. W. Greene, John Wiley and Sons 1981).

"Chelating agents K' of the general formula II J are generally prepared as known from literature by reacting 2-substituted 1,3-propane diamines with benzaldehydes osubstituted with an additional carboxylic or sulphonic 20 acid residue, and optionally successive reaction of the Schiff reagents thus formed with the respective amines and, possibly, separation of the existing protective groups; or by reacting substituted malonic acid halogenides optionally with a benzylamine o-substituted 25 with an additional carboxylic or sulphonic acid residue, as described in the European Patent Applications EP 0 417 870, EP 0 502 594, and EP 0 502 595.

Chelating agents K' of the general formula II K are prepared as known from literature.

Chelating agents K' of the general formula II L are prepared according to the method known to a person skilled in the art, as, for example, described in the European Patent Application EP 0 512 661.

TD938EN.DOC Chelating agents K' of the general formula II M are prepared according to the methods of solid-phase peptide synthesis known from literature (Barany and Marrifield, The Peptides: Analysis, Synthesis, Biology. Academic Press, New York, 1980; Stewart and Young, Solid-phase Peptide .Synthesis, 2nd ed., Pierce Chemical Co., Rockford,' IL, 1984). Cysteines are used with the desired S-protective group. The peptides of formula II M are separated from the resin in an N-protected form. The free carboxyterminal end is activated by a method known to a person skilled in the art, for example, according to the carbodiimide method'(Fieser, Reagents for Organic Synthesis 10, 142), or using a mixed or cyclic anhydride (Org. Prep. Proc. Int. 1975, 2, 215) br an activated ester (Adv. Org. Chem. Part B, 472) Cysteine-rich amino acid sequences of metallothionines (see Patent Application WO 91/17173) are prepared according to the methods of solid-phase peptide synthesis 20 known from literature (Barany and Marrifield, The Peptides: Analysis, Synthesis, Biology. Academic Press, New York, 1980; Stewart and Young, Solid-phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL, 1984). Cysteines are used in S-protected form. The 25 cysteine-rich amino acid sequences are separated from the resin in an N-protected form. The free carboxyterminal end is activated by a method known to a person skilled in the art, for example, according to the carbodiimide method (Fieser, Reagents for Organic Synthesis 10, 142), or using a mixed or cyclic anhydride (Org. Prep. Proc.

Int. 1975, 1, 215) or an activated ester (Adv. Org. Chem.

Part B, 472). The protective groups are removed after coupling to E' according to the methods known to a person skilled in the art from literature, for example, using basic hydrolysis, reductive decomposition, etc.

ID938EN.DOC (see, for example, "Protective Groups in Organic Synthesis", T. W. Greene, John Wiley and Sons 1981).

The present invention further provides agents for diagnosis characterized by the content of a compound complex of the general formula containing metal ions with atomic numbers 21-31, 37-39, 42-51, and 57-83. These agents are suited for various diagnostic methods by choosing the appropriate metal ion.

If the agent according to the invention is meant for use in radiodiagnostics, the central ion of the complex salt o@ must be radioactive. This applies in particular to the ions of elements having atomic numbers 27, 29, 30-32, 37- S 15 39, 42-51, 62, 64, 70, 75, and 77. Preferred isotopes are, for example, 99 mTc, 1 8 6Re, and 1 1 1 In. Endotheline S* derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists may also be radiolabelled using iodine isotopes. 1231 is, for 20 example, a preferred isotope for this use.

If the agent according to the invention is meant for use in NMR diagnostics, the central ion of the complex salt must be paramagnetic. This applies, in particular, to the 25 divalent and trivalent ions of elements having atomic 5* numbers 21-29, 42, 44, and 57-70. Suitable ions are, for example, the chromium(III), manganese(II), iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III), and ytterbium(III) ions.

The gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), and iron(II) ions are particularly preferred due to their strong magnetic moment.

If the agent according to the invention is meant for use in radiodiagnostics, the central ion must derive from an ID938OE.DOC element having a higher atomic number to achieve a sufficient absorption of the X-rays. It has been found that agents for diagnosis are suited for this that contain a physiologically acceptable complex salt with central'ions from elements having atomic numbers between 21-29, 42, 44, 57-83; among these are, for example, the lanthan(III) ion and the abovementioned ions of the lanthanoid series.

If the agent according to the invention is meant for use in positron emission tomography, the central ion must be a positron-emitting isotope. This applies, in particular, to isotopes such as 43Sc, 44 Sc, 52 Fe, 55 Co, 68 Ga and Cu o*to (Heiss, W. Phelps M. Positron Emission Tomography 15 of Brain, Springer Verlag Berlin, Heidelberg, New York 1983).

Another object of the present invention are methods for preparing the agents for diagnosis according to the 20 4nvention.

'h'e radiopharmaceutic agents according to the invention are prepared in a way known in the art by dissolving or suspending the complexing agents according to the inven- 25 tion and their conjugates while optionally adding the additives common in galenics in a hydrous medium and then lyophilizing or sterilizing the solution or suspension optionally. Suitable additives are, for example, physiologically safe buffers (such as tromethamine), auxiliary ligand additives (such as sodium citrate or sodium tartrate), reductants (such as tin(II) chloride), or if need be electrolytes such as sodium chloride, or if need be (one of) the auxiliary agent(s) commonly used in galenics (such as lactose, methyl cellulose, mannitol) and/or tenside(s) (for example, lecithines, Tween®, Myrj®). The composition of ID938EN.DOC the additives used must permit the preparing of the compounds according to the invention.

When applied in vivo in nuclear medicine, the agents according to the invention are dosed at 1 x 10- 5 to 5 x 104 nmol/kg body weight, preferably at quantities between 1 x 10- 3 to 5 x 10 2 nmol/kg body weight. Assuming a medium body weight of 70 kg, the amount of radioactivity for diagnostic applications is between 0.05 and 50 mCi, prefera±Ly 5 to 30 mCi, per application. Normally, a solution of the agent according to the invention is applied as an intravenous, intra-arterial, or peritoneal injection of 0.1 to 5 ml of the agent according to the :io invention. Intravenous application is preferred. Details 15 of its handling and dosage are described, for example, in "Radiotracers for Medical Applications", CRC press, Boca Raton, Florida. The compounds according to the invention are used for radiodiagnostic and radiotherapeucic purposes in the form of their complexes with 20 radioisotopes from elements having atomic numbers 27, 29, 30-32, 37-39, 42-51, 62, 64, 70, 75, and 77.

*The radiopharmaceutic agents according to the invention meet the varied conditions for being used as radiopharmaceutic agents in radiodiagnostics and radiotherapy. They are excellently capable of accumulating in target tissues after i.v. application, thereby permitting a non-invasive diagnosis of said tissues. Water solubility of the radiopharmaceutic agents is ensured if need be as described above by auxiliary agents common in galenics.

Moreover, the radiopharmaceutic agents according to the invention show not only a high in-vitro stability, but also a surprisingly high in-vivo stability, so that the radionuclide bound in the complex is either not released IDJ38EN.DOC or replaced at all, or not to a clinically relevant extent.

The pharmaceutic agents for NMR and radiodiagnostics according to the invention are also prepared in a known way by dissolving or suspending the complex compounda according to the invention in a hydrous medium while optionally adding, the additives common in galenics and then optionally sterilizing the solution or suspension.

Suitable additives are, for example, physiologically safe buffers (such as tromethamine), complexing agent additives (such as diethylene triamine pentaacetic acid), or if need be electrolytes such as sodium chloride or if need be antioxidants such as ascorbic acid.

The pharmaceutic agents according to the invention used in NMR diagnostics contain preferably lmol to 1 mol/1 of complex salt and are normally applied at dosages of 0.001 to 5 mmol/kg. They are meant for enteral and parenteral 20 application. The compound complexes according to the invention are used for NMR and radiodiagnostic purposes in the form of their complexes with ions of elements having atomic numbers 21-29, 42, 44, and 57-83.

25 The agents according to the invention meet the varied conditions required for their use as contrast media in nuclear spin tomography. After oral or parenteral application, they increase signal intensity and are thus excellently suited for improving the meaningfulness of an image obtained by nuclear spin tomography. Moreover, they are highly effective, which minimizes the amount of foreign matter the human body has to deal with, and well compatible to ensure the non-invasive nature of the examinations.

ID38BN.DOC Neutralization is done by inorganic bases (such as hydroxides, carbonates or bicarbonates) of, for example, sodium, potassium, lithium, magnesium, or calcium and/or of organic bases such as primary, secondary, and tertiary amines like ethanol amine, morpholine, glucamine, Nmethyl and N,N-dimethyl glucamine, as well as basic amino acids like lysine, arginine, and ornithine, and of amides of originally neutral or acidic amino acids.

The agents according to the invention are excellently suited for use as x-ray contrast medium; it should be pointed out, in particular, that biochemical and pharmacological tests did not reveal any signs of immediate allergy reactions as they are known from other 15 contrast media. They are of particular value for digital subtraction techniques due to their favourable absorbing capabilities in higher tube voltage ranges.

The pharmaceutic agents according to the invention are 20 further prepared in a way known in the art by dissolving the complexing agents according to the invention while adding a reducing agent, preferably tin(II) salts such as chloride or tartrate, and, optionally the additives common in galenics in a hydrous medium and then sterilizing them by filtration. Suitable additives are, :for example, physiologically safe buffers (such as tromethamine), minor quantities of electrolytes (such as sodium chloride), stabilizers (such as gluconate, phosphates, or phosphonates). The pharmaceutic agent is either present as a solution or in lyophilized form; briefly before its application it is mixed either with a Tc-99m pertechnetate solution eluted from commercially available generators, or with a perrhenate solution.

A cold kit according to the invention is provided to prepare the radiopharmaceuticals. It contains a compound ID938N.DOC according to the invention of the general formula a reductant, and, optionally, one or more auxiliary ligands, either in solution, dry, or lyophilized. The cold kit, moreover, contains directions for use including directions for reacting the compounds according to the invention of general formula with a permetallate of a radioactive metal ion, whereby a complex according to the invention is formed containing the compound of general formula and the metal ion.

Furthermore, a cold kit is provided to prepare the radiopharmaceuticals that contains endothelines, .endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists bonded 15 to a peptide, a derivative, or a chelating agent capable of binding metal atoms and, optionally, a metal atom bonded to it. This latter metal atom may not be radioactive and can easily be replaced by a radioactive isotope.

Another object of the present invention is a hot kit containing endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists bonded to a peptide, a derivative, or a chelating agent with a radioactive iodine atom bonded to the latter.

*Soo: Another object of the present invention is the use of the cold kit for preparing a radioactive formulation comprising a closeable vessel containing a predetermined quantity of endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists bonded to a peptide, a derivative, or a chelating agent capable of binding metal atoms, and containing a quantity of a reducing agent sufficient to label the compound with 99 mTc.

ID93 8N.DOC Another object of the invention is a method for imaging pathological vascular changes characterized in that a complex of a compound of general formula containing metal ions of atomic numbers 21-32, 37-39, 42-51, and 57- 83 is used as a contrast medium.

The method according to the invention for imaging pathological vascular changes is further characterized in that a radiolabelled endotheline is used as a contrast medium. The radiolabelled endotheline used in the method according to the invention for imaging pathological vascular changes preferably contains a radioactive iodine isotope. 131I, 125I, and 1231 are particularly preferred.

15 These iodinated endothelines are prepared in a generally known manner, preferably according to the chloramine-T method, the lactoperoxidase method, the Bolton-Hunter method, or the iodogen method.

20 In one method of carrying out a radiodiagnostic examination, the radiopharmaceutic composition is applied to a patient at a dose of 0.1 to 30 mCi, preferably of 0.5 to 10 mCi per 70 kg of body weight, and the radiation emanating from the patient is recorded.

S* Figures 1 and 2 illustrate the method according to the invention for imaging pathological vascular changes.

Fig. 1 shows a posterior planar recording of a WHHL rabbit 5 h p.i. of 123I endotheline 1.

Fig. 2 shows a Sudan(III) dyeing and an autoradiograph of the aorta of a WHHL rabbit. The aorta was removed 5 h p.i. of 123I endotheline 1.

ID938EN.DOC The object of the invention is described in more detail by the following examples.

Example 1 In-vivo and in-vitro accumulation of 1231 endotheline 1 in WHHL rabbits.

Endotheline 1 was labelled with 123I using the chloramine-T method (Krohn et al. 1977, BBA 490, 49). pl endotheline 1 (2 pg, equivalent to 0.8 nmol, in phosphate buffer, 0.25 mol/l, pH 7.4; SIGMA Co.) are added to 50 pA phosphate buffer (0.25 mol/l, pH 100 1 of 123I (15 il 1231, Medgenix Co., 74 MBq, equivalent 15 to 2 mCi, brought to a total volume of 100 Al by adding 0.01 mol/l of NaOH), and 100 p1 chloramine-T solution (35.5 mmol/l phosphate buffer, 0.25 mol/l, pH 7.4) for 2 mins at room temperature. 1000 il sodium disulphite solution (Na 2

S

2 0 5 6.3 mmol/l, 0.25 mol/l, pH 7.4) are 20 added and briefly mixed to stop the reaction. 800 pl sodium iodide solution (13.3 mmol/l phosphate buffer, 0.25 mol/l, pH 7.4) are pipetted into the initial charge to saturate the still vacant valencies (total charge *quantity 2070 il). This reaction charge is gel filtrated through Sephadex G-10 (mobile buffer: phosphate buffered saline 0.1 BSA Labelling of endotheline e was successful at 39 The five fractions of 1231 endotheline 1 with the highest activitywere collected (4.5 ml) and applied to the anaesthesized WHHL rabbit (Rompun/Ketavet 1:2) via an ear vein. Due to the missing or defective LDL receptor, WHHL rabbits have a high LDL level in their blood and thus spontaneously show atherosclerotic vascular changes. The dose applied was equivalent to 0.2 mmol 123I endotheline 1 with an activity of 13.69 MBq (0.37 mCi), and a ID938EN.DOC specific activity of 68.5 MBq/mmol. During the test period of 5 h after the application, static scintigraphs were made from various positions using a gamma camera (Elcint SP4 HR). The rabbit was killed 5 h after application, and an autoradiograph of the aorta and Sudan III dyeing were carried out. The aorta of the WHHL rabbits could be imaged in vivo 4 h p.i. due to the labelling of endotheline with 123I (see Fig. A subsequent autoradiography revealed a complete labelling of the aorta wall and the atherosclerotic plaques (see Fig. The enrichment factor between normal and atherosclerotic wall regions was between 8 and 12 depending on the formation of the plaques (Sudan III dyeing) Example 2 a) S-benzoylthioacetyl-gly-gly-gly-gly-asp-his-leu-aspile-ile-trp.

A solution of 50 mg S-benzoylthioacetyl-gly-gly-gly and 16 mg N-hydroxysuccinimide in absolute freshly distilled dimethyl formamide is cooled to -15°C and added to 29 mg dicyclohexyl carbodiimide in absolute dimethyl formamide.

The reaction mixture is stirred for 2 hours at -5 0 C, then for 2. hours at room temperature, and then cooled to 150C. N,N'-dicyclohexyl urea that settles down is filtered off. The filtrate is added to a solution of 1 mg gly-asp-his-leu-asp-ile-ile-trp in absolute dimethyl formamide and stirred for 20 hours at room temperature.

The reaction solution is concentrated to a minimum under vacuum. Dropping in diethyl ether results in a flocculent precipitate which is isolated and then purified using preparative HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the ID938E.DOC organic solvent portion is blown of f using N 2 and the residue is freeze-dried.

Molecular weight: calc. 1301.4 det. 1301 (FAB-MS) b) 99 mTc complex of S-benzoylthioacetyl-gly-gly-glygly-asp-his-leu-asp-ile-ile-trp.

A solution of 0.5 mg of S-benzoylthioacetyl-gly-gly-glygly-asp-his-leu-asp-ile-ile-trp in 300 p.1 phosphate buffer (Na 2

HPO

4 0.1 mol/l, pH 9.5) is added to 50 p.1 of a 0.15 molar trisodium citrate dihydrate solution and p.1 of a 0.2 molar tin(II) chloride dihydrate solution.

The reaction mixture is added to a pertechnetate solution (0.4-0.9 mCi) from a Mo-99/Tc-99m generator, incubated 4 for 15 min at room temperature and then filtrated (0.2 p.

filter) Labelling is analysed using HPLC: MERCK nucleosil-column, 125 x 4 mm, 5 pVm; gradient from 100 !k A to 100 t B within 7.5 min. Eluent A: phosphate buffer 20 (Na 2

HPO

4 0.01 M; pH 2.0) eluent B:acetonitrile/ phosphate buffer (Na 2

HPO

4 0. 01 M; pH 2. 0) 75:25 flow rate: 1.0 ml/min.

Example 3 a) N,N' -bis (S-benzoylthioacetyl) -3,4-diaminobutyrylcys-ser-cys-ser-ser-leu-met -asp-lys-glu-cys-val -tyrphe-cys-his-leu-asp-ile-ile-trp, A solution of 50 mg N,N'-bis(S-benzoylthioacetyl)-3,4diaminobutyric acid and 15 mg N-leydroxysuccinimide in absolute freshly distilled dimethyl formamide is cooled to -15 0 C and added to 28 mg dicyclohexyl carbodiimide in absolute dimethyl formamide. The reaction mixture is WD93flKN.DOC stirred for 2 hours at -5 0 C, then for 2 hours at room temperature, and then cooled to -15°C. N,N'-dicyclohexyl urea that settles down is filtered off. The filtrate is added to a solution of 1 mg cys-ser-cys-ser-ser-leu-metasp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp (endotheline 1) in absolute dimethyl formamide and stirred for 20 hours at room temperature. The reaction solution is concentrated to a minimum under vacuum.

Dropping in diethyl ether results in a flocculent precipitate which is isolated and then purified using HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freezedried.

Molecular weight: calc. 2948.5 det. 2949 (FAB-MS) b) 9 9 mTc complex of 20 N,N'-bis(S-benzoylthioacetyl)-3,4-diaminobutyryl- 6 I I cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyrphe-cys-his-leu-asp-ile-ile-trp, N,N'-bis(S-benzoylthioacetyl)-3-4-diaminobutyryl-cys-sercys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-hisleu-asp-ile-ile-trp is labelled according to example 2b.

Example ,4 a) Amide conjugate of 4-carboxyethylphenylglyoxal-bis- (N-methylthio semicarbazone)-N-hydroxy succinimide ester and I cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-

I

ID9381N.DOC cys-his-leu-asp-ile-ile-trp.

A solution of 1, mg 4-carboxyethylphenylglyoxal-bis- (Nmethylthio semicarbazone)-N-hydroxy succinimide ester in 50 gl dioxane is added to a solution of 1 mg cys-ser-cysser-ser-trp-leu-asp-lys-glu-.cys-val -tyr--phe-cys-his-leuasp-ile-ile-trp (endotheline 2) in 100 jil phosphate buffered saline, pH 8.4, in a nitrogen atmosphere, and stirred for 15 hours at a temperature below 10 0 C. The conjugate is purified using a preparative HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freeze-dried.

Molecular weight: calc. 2895.4 det. 2894 (FAB-MS) b) 9 9 mTc complex of 3- (p-phenylglyoxal-di (N-methylthio semicarbazone)] propionyl-cys-ser-cys-ser-ser-trp- leu-asp-lys-glu-cys- ::0val -tyr-phe-cys-his-leu-asp-ile-ile-trp.i able according to example 2b.

Example a) 3,6-Diaza-1,8-dimercapto-2,7-bis- (carbonyl-gly-hisleu-asp-ile-ile-trp) octane.

WSJBERN.DOC

256 mg (0.5 mmol) N,N'-1,2-ethylene diyl-bis-L-cysteinedi-(4-nitrophenyl) ester (manufactured according to EP 0 279 417) is added in portions in an argon atmosphere to a solution of 853 mg (1 mmol) NH 2 -gly-his-leu-asp-ile-iletrp (manufactured in analogy to Barany and Marrifield, The Peptides: Analysis, Biology, Academic Press, New York, 1980; Stewart and Young, Solid-Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL, 1984) and 304 mg (3 mmol) triethyl amine in 100 ml absolute dimethyl formamide. The mixture is stirred for 24 h at room temperature. When the reaction is finished, the mixture is filtrated, and the solvent is drawn off under vacuum. To the remaining oil 50 ml diethyl formamide is added and evaporated three times. The 15 residue is mixed up with 200 ml absolute diethyl ether.

The white solid that settles down is filtered off. It is recrystallized from dimethyl formamide/diethyl ether mixtures.

20 Yield: 278 mg (28.7 white powder.

Analysis: calculated on the anhydrous substance Calc.: C 55.77 H 6.86 N 15.90 0 18.16 S 3.31 S•Found: C 55.49 H 7.03 N 15.81 S 3.07 b) 99 mTc complex of 3,6-diaza-1,8-dimercapto-2,7-bis- (carbonyl-gly-his-leu-asp-ile-ile-trp) octane.

A solution of 0.5 mg of the prepared 3,6-diaza-l,8dimercapto-2,7-bis-(carbonyl-gly-his-leu-asp-ile-ile-trp) octane in 300 gl phosphate buffer (Na 2

HPO

4 0.5 mol/1, pH is added to 50 gl of a 0.15 molar trisodium citrate dihydrate solution and 2.5 .l of a 0.2 molar tin(II) chloride dihydrate solution. The reaction mixture is added to a pertechnetate solution (0.4-0.9 mCi) from a Mo-99/Tc-99m generator, incubated for 10 min at room temperature and then filtrated (0.2 p filter). Labelling ID938EN.DOC is analysed using HPLC: MERCK nucleosil-column, 125 x 4 mm, 5 j Lm; gradient from 100 k A to 100 k B within min. Eluent A: phosphate buffer (Na 2

HPO

4 0. 01 M; pH 50 50 flow rate: 1.0 ml/min.

Example 6 a) {[(3,3,9,9-tetramethyl-4,8-diaza-undecyl-2,l0-diondioxime) -6-yl]methylphen-41*-yl} aminothiocarbonyl-cys- 7 ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cyshis-leu-asp-ile-ile-trp.

A solution of 1 mg 6-(4'-isothiocyanatobenzyl)-3,3,9,9- 15 tetramethyl-4,8-diazaundecane-2,10-dion-dioxime in 50 Ll tetrahydrofurane is mixed in a nitrogen atmosphere with a solution of 1 mg cys-ser-ala-ser-ser-leu-met-asp-lys-gluala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp (alaendotheline) in 100 lil water and stirred overnight. The conjugate is purified using a preparative HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freeze-dried.

Molecular weight: calc. 2833.3 det. 2833 (FAB-MS) b) S9 9 mTc complex of {[(3,3,9,9-tetramethyl-4,8-diazaundecyl-2, lo-dion-dioxime) -6-yl]methylphen-4' -yl} aminothiocarbonyl-cys-ser-ala-ser-ser-leu-met-asp-lysglu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp.

9-tetramethyl-4, 8-diaza-undecyl-2, lO-diondioxime) -6 -yl]methylphen-4 yl} aminothiocarbonyl-cysser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys- 1D9 3 1N.DOC his-leu-asp-ile-ile-trp is labelled according to example 2b.

Example 7 a) pentyl-cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-

LI

tyr-phe-cys-his-leu-asp-ile-ile-trp.

A solution of 50 mg diaminopentanoic acid and 15 mg N-hydroxy succinimide in absolute freshly distilled dimethyl formamide is cooled 0 15 to -15 0 C and added to 28 mg dicyclohexyl carbodiimide in absolute dimethyl formamide. The reaction mixture is stirred for 2 hours at then for 2 hours at room temperature, and then cooled to -15°C. N,N'-dicyclohexyl urea that settles down is filtered off. The filtrate is 20 added to a solution of 1 mg cys-ser-cys-ser-ser-leu-metasp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp (endotheline 1) in absolute dimethyl formamide and stirred for 20 hours at room temperature. The reaction solution is concentrated to a minimum under vacuum.

Dropping in diethyl ether results in a flocculent precipitate which is isolated and then purified using preparative HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freeze-dried.

Molecular weight: calc. 2962.5 det. 2963 (FAB-MS) ID93GEN.DOC b) 99 mTc complex of diamino-l-oxo-pentyl-cy.--ser- cys-ser-ser-leu-met-asply-licsvltrpecshsluapieietp N,N' -bis(S-benzoylthioacetyl) cys-ser-cys-ser-ser-leu-et-asp-ys-glu-cys-val-yrphecys-his-leu-asp-ile-ile-trp is labelled according to example 2b.

Example 8 a) {[2,6-diaza-1,7-di(2-hydroxyphenyl) -4-methyl-hept-4-yl] -methyl -phen-4' -yl} aminothiocarbonylcys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyrtyr-cys--his-leu--asp-ile-ile-trp.

A solution of 1 mg 2-methyl-2-(4-isothiocyanatobenzyl)- ~'20 N,N'propylene-bis-salicylidenamine in 50 A.l tetrahydrofurane is mixed in a nitrogen atmosphere with a solution of 1 mg cys-thr-cys-phe-thr-tyr-lys-asp-lys-glucys-val-tyr-tyr-cys-his-leu-asp-ile-ile-trp (endotheline 3) in 100 j[il water and stirred overnight. The conjugate is purified using a preparative HPLC (gradient: CC acetonitrile/phosphate buffer). After neutralizing the :~-:buffered solution, the organic solvent portion is blown of f using N 2 and tha residue is freeze-dried.

Molecular weight: calc. 3090.7 det. 3091 (FAB-MS) b) 99 m Tc complex of 6-diaza-l, 7-di (2-hydroxyphenyl) 4-ehlhp--l-mty-hn4-l aminothiocarbonylcys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val -tyr-tyrcys-his-leu-asp-ile-ile-trp.

Ifl93SEEN.DOC {[2.6-diaza-1,7-di(2-hydroxyphenyl) -4-rnethyl-hept-4-y.]methyl-phen-4'-yll aminothio-carbonyl-cys-thr-cys-phethr -tyr-lys-asp-lys-glu-cys-val-tyr-tyr-cys-his-leu-asp.

ile-ile-trp is labelled according to example 2b.

Example 9 a) (2,6-diaza-1,7-di(2-hydroxy-5-sulphophenyl) -4methyl-hept-4-yl]-methylphen-4' -yl} aminothiocarbonylala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala- val-tyr-pheala-his-leu-asp-ile-ile-trp.

A fresh solution of 1 mg 2-methyl-2-(4isothiocyanatobenzyl) -propylene-bis- :9..:(sulpho)salicylidenamine] in 50 jil water is mixed in a 9 9*nitrogen atmosphere with a solution of 1 mg ala-ser-alaser-ser-leu-met -asp-lys-glu-ala-val-tyr-phe-ala-his-leuasp-ile-ile-trp) in 100 p1l water and stirred overnight.

20 The conjugate is purified using a preparative HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freeze- 99999dried.

SMolecular weight: calc. 2959.4 9det. 2959 (FAB-MS) b) 9 9 m"Tc complex of {[2,G-diaza-1,7-di(2-hydroxy-5sulphophenyl) -4-methyl-hept-4yl]-methylphen-4' -yl} aminothiocarbonyl-ala-ser-ala-serser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-aspile-ile-trp.

6-diaza-1,7-di(2-hydroxy-5-sulphophenyl) -4-methylhept -4 -yl]-methylphen-4 yl} aminothiocarbonyl-ala-ser- 1D93SEN.DOC ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-hisleu-asp-ile-ile-trp is labelled according to example 2b.

Example a) N-[3,6,9-triaza-l-oxo-3,6,9-tris- (hydroxycarbonylmethyl)-9-ethoxycarbonylmethyl)-nonyl]gly-his-leu-asp-ile-ile-trp.

403.4 mg (1 mmol) 3-ethoxycarbonylmethyl-6-[2-(2,6dioxomorpholino)-ethyl]-3,6-diaza-octanedioic acid (DTPAmonoethyl ester monoanhydride) is added in portions to a solution of 853 mg (1 mmol) NH 2 -gly-his-leu-asp-ile-ileo. trp (prepared in analogy to Barany and Marrifield, The 15 Peptides: Analysis, Biology, Academic Press, New York, 1980; Stewart and Young, Solid-Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL, 1984) and 506 mg (5 mmol) triethyl amine in 100 ml absolute dimethyl formamide. The mixture is stirred for 24 h at room 20 temperature. When the reaction is finished, the mixture is filtrated, and the solvent is drawn off under vacuum.

To remaining oil 50 ml diethylformamide is added and *l*s evaporated three times. The residue is mixed up with 200 t ml absolute diethyl ether. The white solid that settles down is filtered off. It is recrystallized from dimethyl formamide/diethyl ether mixtures.

0 Yield: 634 mg (50.4 white powder.

Analysis: calculated on the anhydrous substance Calc.: C 54.49 H 6.82 N 14,49 0 24.20 Found: C 54.23 H 7.03 N 14.34 b) Synthesis of N-[3,6,9-triaza-1-oxo-3,6,9,9-tetra- (hydroxycarbonylmethyl)-nonyl]-gly-his-leu-asp-ile-iletrp.

ID938EH.DOC 1.26 mg (1 mmol) of the prepared N-[3,6,9-triaza-l-oxo- 3,6,9-tris-(hydroxycarbonyl-methyl)-9ethoxycarbonylmethyl)-nonyl]-gly-his-leu-asp-ile-ile-trp (example 10a) is suspended in 100 ml water. A pH value of 12.5 is set by adding 10 molar, hydrous sodium hydroxide solution. The mixture is stirred for 5 h at room temperature, and a pH value of 2 is set by adding concentrated hydrochloric acid. The crude conjugate is filtered off and rewashed using a small amount of iced water and ice-cooled methanol. For purifying, it is taken up in little water at pH 7 and chromatographed on silica gel RP-18 (eluens: water/tetrahydrofurane, tetrahydrofurane: 0-50 The fractions containing the 15 conjugate are evaporated and the residue is taken up in a small amount of water at pH 4.5. The product is precipitated at pH 2, achieved by adding concentrated hydrochloric acid, and dried under reduced pressure.

20 Yield: 213 mg (17.3 white powder.

Analysis: calculated on the anhydrous substance Calc.: C 53.78 H 6.65 N 14,82 0 24.75 Found: C 53.49 H 6.91 N 14.57 Alternatively, N-[3,6,9-triaza-1-oxo-3,6,9,9-tetra- (hydroxycarbonylmethyl)-nonyl]-gly-his-leu-asp-ile-iletrp (example 10b) can also be prepared by reacting 853 mg (1 mmol) NH 2 -gly-his-leu-asp-ile-ile-trp (example dissolved in 100 ml water, at a pH of 9 in portions with 3.57 g (10 mmol) bis-(aminoethyl)-N,N,N',N",N'"pentaacetic acid dianhydride. The mixture is stirred for 3 h at room temperature while permanently keeping the pH value of the reaction medium at 9 by dropping in 1N hydrous sodium hydroxide solution. Then, a pH value of 2 is set by adding concentrated hydrochloric acid. The crude conjugate is filtered off and rewashed using a ID938 N.COC small amount of iced water and ice-cooled methanol. For purifying, it is taken up in a small amount of water at pH 7 and chromatographed on silica gel RP-18 (eluens: water/tetrahydrofurane, tetrahydrofurane: 0-50 The fractions containing the conjugate are evaporated and the residue is taken up in a small amount of water at pH The product is precipitated at pH 2, achieved by adding concentrated hydrochloric acid, and dried under reduced pressure.

Yield: 317 mg (25.7 white powder.

c) 9 9 mTc complex of N-[3,6,9-triaza-l-oxo-3,6,9,9-tetra- (hydroxycarbonylmethyl) -nonyl]-gly-his-leu-asp-ile-ile- 15 trp A solution of 0.5 mg of the prepared N-[3,6,9-triaza-loxo-3,6,9, 9-tetra- (hydroxy-carbonylmethyl) -nonyl]-glyhis-leu-asp-ile-ile-trp (example 10b) in 300 gl phosphate 20 buffer (Na 2

HPO

4 0.5 mol/l, pH 7.0) is added to 50 .l of a 0.15 molar trisodium citrate dihydrate solution and pl of a 0.2 molar tin(II) chloride dihydrate solution.

S• The reaction mixture is added to a pertechnetate solution (0.4-0.9 mCi) from a Mo-99/Tc-99m generator, incubated fcr 10 min at room temperature and then filtrated (0.2 u filter).,Labelling is analysed using HPLC: MERCK nucleosil-coulmn 125 x 4 mm, 5 pm; gradient from 100 A to 100 B within 7.5 min. Eluent A: phosphate buffer (Na 2

HPO

4 0.01 M; pH 2.0) 50 50 flow rate: ml/min.

d) 11 1In complex of N-[3,6,9-triaza-l-oxo-3,6,9,9-tetra- (hydroxycarbonylmethyl) -nonyl]-gly-his-leu-asp-ile-iletrp ID938wH.DOC 62 A solution of 0.5 mg of the prepared N-[3,6,9-triaza-loxo-3,6,9,9-tetra- (hydroxy-carbonylmethyl) -nonyl]-glyhis-leu-asp-ile-ile-trp (example 10b) in 300 ji phosphate buffer (Na 2 HP0 4 0.5 mol/l, pH 6.5) is incubated for min at room temperature using 20 l 1 11 InC13 (3 MBq, NEN, Du Pont) and then filtrated (0.2 j filter). Labelling is analysed using HPLC: MERCK nucleosil-column 125 x 4 mm, jim; gradient from 100 A to 100 B within 7.5 min.

Eluent A: phosphate buffer (Na 2 HP0 4 0.01 M; pH 2.0) 50 flow rate: 1.0 ml/min.

e) Gd(III) complex of N-[3,6,9-triaza-l-oxo-3,6,9,9tetra- (hydroxycarbonylmethyl) -nonyl]-gly-his-leu-asp-ileile-trp, sodium salt.

669 mg (2 mmol) gadolinium(III) acetate is added in portions at a pH of E.5 to a solution of 2.46 g (2 mmol) of the prepared N-[3,6,9-triaza-l-oxo-3,6,9,9-tetra- (hydroxy-carbonylmethyl)-nonyl]-gly-his-leu-asp-ile-ile- 20 trp (example 10b) in 100 ml water while keeping the pH of the reaction mixture between 6 and 6.5 by adding 10 N of hydrous sodium hydroxide solution. When the total amount of gadolinium has been added, the mixture is stirred for 4 h. After complexing has been finished, the product is set to a pH value of 7 by dropping in 1 N of hydrous hydrochloric acid solution and filtrated. The clear solution is freeze-dried. The residue is taken up in a small amount of water and chromatographed on silica gel RP-18 (eluens: water/tetrahydrofurane, tetrahydrofurane: 0-50 The fractions containing the product are freed from tetrahydrofurane using a rotary evaporator; the residual solution is freeze-dried.

Yield: 1.13 mg (39.6 white powder.

Analysis: calculated the anhydrous substance ID9381.DOC Calc.: C 46.31 H 5.37 N 12.76 0 21.31 Gd Na 3.22 11.02 Found: C 46.09 H 5.63 N 12.49 Gd Na 3.51 10.83 Example 11 Gd(III) complex of 1-{2-hydroxy-3- [4-(gly-his-leu-aspile-ile-trp-thiouridyl)phenoxy] propyl} 1,4,7,10tetraaza-4,7,10-tris-(carboxylatomethyl)-cyclododecane 708 mg (1 mmol) 1-[2-hydroxy-3-(4-isothiocyanatophenoxy)-propyl]-1,4,7,10-tetraaza-4,7,10-tris- 10 (carboxylatomethyl) cyclododecane, Gd complex (manufactured according to EP 0 485 054) is added in portions in an argon atmosphere to a solution of 853 mg (1 mmol) NH 2 -gly-his-leu-asp-ile-ile-trp (prepared in analogy to Barany and Marrifield, The Peptides: Analysis, Biology, Academic Press, New York, 1980; Stewart and Young, Solid-Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL, 1984) and 304 mg (3 mmol) triethyl amine in 100 ml absolute dimethyl formamide. The mixture is stirred for 24 h at room temperature. When the 20 reaction is finished, the mixture is filtrated, and the solvent is drawn off under vacuum. To the remaining oil 50 ml diethyl formamide is added and evaporated three times. The residue is mixed up with 200 ml absolute diethyl ether. The white solid that settles down is filtered off. It is recrystallized from dimethylformamide/diethyl ether mixtures.

Yield: 533 mg (34.0 white powder.

Analysis: calculated on the anhydrous substance Calc.: C 50.02 H 5.94 N 13.46 O 18.45 S 2.05 Gd 10.07 ID938sH.DOC Found: C 49.78 H 6.23 N 13.17 S 1.89 Gd 9.83 Example 12 a) cys(acm)-gly-cys(acm)- I I cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-

L-_

phe-cys-his-leu-asp-ile-ile-trp.

A solution of 50 mg Fmoc-cys(acm)-gly-cys(acm) and 15 mg N-hydroxy succinimide in absolute, freshly distilled dimethylformamide is cooled to -15 0 C and added to 28 mg dicyclohexyl carbodiimide in absolute dimethylformamide.

15 The reaction mixture is stirred for 2 hours at -50C, then for 2 hours at room temperature, and then cooled to 150C. N,N'-dicyclohexyl urea that settles down is filtered off. The filtrate is added to a solution of 1 mg cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe- 20 cys-his-leu-asp-ile-ile-trp (endotheline 1) in absolute dimethylformamide and stirred for 20 hours at room temperature. To split off the protective group, some piperidine is added to the reaction solution, stirred for min, and subsequently concentrated to a minimum under 25 vacuum. Dropping in diethyl ether results in a flocculent precipitate which is isolated by centrifugation and then purified using preparative HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freeze-drzed.

Molecular weight: calc. 2897.4 det. 2898 (FAB-MS) ID938EH.DOC b) 99 mTc complex of cys(acm)-gly-cys(acm)- I- I cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr- Ll phe-cys-his-leu-asp-ile-ile-trp.

Cys(acm)-gly-cys(acm)-cys-ser-cys-ser-ser-leu-met-asplys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp is labelled according to example 2b.

Example 13 a) Cyclo(trp-leu-val-pro-asp)-cys(acm)-gly-cys(acm).

15 A solution of 133.0 mg Cyclo(trp-leu-val-pro-asp) (prepared according to EP 0,436,189) and 41.3 mg hydroxybenzotriazole in 6 ml absolute, freshly distilled dimethylformamide is cooled to -150C and added to a solution of 51.7 mg 1-ethyl-3-(3-dimethylaminopropyl) 20 carbodiimide hydrochloride in 8 ml absolute dimethyl formamide. The reaction mixture is stirred for 2 hours at then for 2 hours at room temperature while slowly dropping in a solution of 134.0 mg cys(acm)-gly-cys(acm)- O-t-but in absolute dimethyl formamide. After stirring on for 7 h, the mixture is concentrated under vacuum to a minimum. Present protective groups are removed using trifluoroacetic acid, the peptide is precipitated by adding ether and then purified using preparative HPLC (gradient: acetonitrile/ phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freezedried.

Molecular weight: calc. 1016.2 det. 1016 (FAB-MS) ID938EN.DOC b) 99 mTc complex of cyclo(trp-leu-val-pro-asp)cys(acm)-gly-cys(acm).

Cyclo(trp-leu-val-pro-asp)-cys(acm)-gly-cys(acm) is labelled according to example 2b.

Example 14 a) 4-mercaptobutyrimidyl-cys-ser-ala-ser-ser-leu-metasp-lys-glu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp A solution of 500 ug cys-ser-ala-ser-ser-leu-met-asp-lysglu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp (ala- 15 endotheline) in 400 pl phosphate buffered saline (pH 7.4) is incubated for one hour at room temperature with a 100 times molar excess of Traut reagent (2.6 mg imino thiolane in 400 p1 triethanolamine hydrochloride buffer pH Then the reaction is finished by separating 20 excessive Traut reagent using a gel filtration column (SEC 3000 Beckman). The reaction is checked by determining the free SH groups (according to Grasseti).

Molecular weight: calc.: 2504.9 det.: 2505 (FAB-MS) 99mTc complex of 4-mercaptobutyrimidyl-cys-ser-alaser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-his-leuasp-ile-ile-trp A solution of 0.5 mg of 4-mercaptobutyrimidyl-cys-serala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-hisleu-asp-ile-ile-trp in 300 p1 phosphate buffer (Na 2

HPO

4 0.1 mol/l, pH 9.5) is added to 50 p1 of a 0.15 molar trisodium citrate dihydrate solution and 2.5 p1 of a 0.2 molar tin(II) chloride dihydrate solution. The reaction ID938EN.DOC 67 mixture is added to a pertechnetate solution (0.4-0.9 mCi) from a Mo-99/Tc-99m generator, incubated for 15 min at room temperature and then filtrated (0.2 p. filter).

Labelling is analysed using HPLC: MERCK nucleosil-column, 125 x 4 mm, 5 tm; gradient from 100 A to 100 B within min. Eluent A: phosphate buffer (Na 2

HPO

4 0.01 M; pH eluent B: acetonitrile/ phosphate buffer (Na 2

HPO

4 0.01 M; pH 2.0) 75:25 flow rate: 1.0 ml/min.

Example a) 3-Thiopropionyl-gly-asp-his-leu-asp-ile-ile-trp.

A solution of 500 pg gly-asp-his-leu-asp-ile-ile-trp in 15 400 gl phosphate buffered saline is added to a 10 times molar excess of N-succinimidyl-3-(2-pyridylthio) propionate in dimethylformamide and incubated for 2 hours at room temperature. The present 2-pyride sulphide group is then reduced using 25 mM dithiothreitol, pH 4.5. The 20 reaction solution is concentrated under vacuum to a 'minimum. Then it is purified using preparative HPLC (gradient: acetonitrile/phosphate buffer). After neutralizing the buffered solution, the organic solvent portion is blown off using N 2 and the residue is freezedried.

9* Molecular weight: calc. 1040.2 det. 1040 (FAB-MS) b) 99 mTc complex of 3-Thiopropionyl-gly-asp-his-leuasp-ile-ile-trp.

3-Thiopropionyl-gly-asp-his-leu-asp-ile-ile-trp is labelled according to example 2b.

ID938OErOC 68 Example 16 a) 2- (Acetylthio) succinyl-gly-asp-his-leu-asp-ile-iletrp.

A solution of 500 jig gly-asp-his-leu-asp-ile-ile-trp in [tl dimethyl forrnamide is added to a 10 times molar excess of 2-mercaptoacetyl succinic acid anhydride in jil dimethylformamide and incubated for 2 hours at room temperature. Then it is purified using preparative IPLC (gradient: acetonitrile/phosphate buffer). The organic solvent portion is blown off using N 2 and the residue is freeze-dried.

Molecular weight: calc. 1126.2 -*see: 15 det. 1126 (FAB-MS) b) 99 mTc complex of 2- (Acetylthio) succinyl-gly-asp-hisleu-asp-ile-ile-trp.

2- (Acetylthio) succinyl-gly-asp-his-leu-asp-ile-ile-trp is labelled according to example 2b.

Example 17 a) Reduction of 4 cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe- Li cys-his-leu-asp-ile-ile-trp (endotheline 1) A soluti6n of 0.5 mg cys-ser-cys-ser-ser-leu-met-asp-lysglu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp in 500 Ill phosphate buffered saline (pH 7.4) is mixed with 18 Iii mercapto ethanol and incubated for 30 min at room temperature; the excessive reductant is separated.

1D938K?4.DOC 69 b) 9 9mTc complex of cys-ser-cys-ser--ser-leu-met-asplys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp A solution of 0.5 mg of the reduced endotheline 1 in 300 pil phosphate buffer (Na 2

HPQ

4 t 0.5 mau/l, PH 9.5) is added to 50 .il of a 0.15 molar trisodium citrate dihydrate solution and 2.5 1jil of a 0.2 molar tin(II) chloride dihydrate solution. The reaction mixture is added to a pertechnetate solution (0.4-0.9 mCi) from a Mo-99/Tc-99m generator, incubated for 10 min at room temperature and then filtrated (0.2 [t filter). Laoelling is analysed using HPLC: MERCK nucleosil-column, 125 x 4 mm, 5 pLm; gradient from 100 A to 100 t B within 7.5 min Eluent A: phosphate buf fer (Na 2

HPO

4 0. 01 M; pH 2. 0) eluent B: ace toni trile /phosphate buffer (Na 2

HPO

4 0.01. M; pH 0 5 50 flow rate: 1. 0 ml /min.

ID9388N.DDC

Claims (3)

1. Complexes of compounds of the general formula (I) E-L-(K)b (I) containing metal ions with the atomic numbers 21-32,

37-39, 42-51, and 57-83, wherein E is a residue derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline 15 antagonists, or a residue derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists carrying free thiol groups and directly binding a metal ion; L is a direct linkage or a Z 1 -R-Z 2 residue, wherein R is a unbranched, branched, saturated or unsaturated C-l 20 -alkyl group, optionally 25 interrupted by one or several oxygen and/or sulphur atoms and/or carbonyl, -NHCO-, -N(C 1 6 alkyl)CO-, -NH- and -N(Ci. 6 alkyl) groups, and optionally substituted with hydroxy and/or epoxy groups; Z 1 and Zz are, independent of each other, an or group, or a residue of formula a ID93BiNA.DOC (CH 2 Z 2 Z, -(CH 2 )s B a wherein s and t are, independent of each other, the integers 0, 1, 2, or 3, ring B represents a phenyl or cyclohexyl group, and Z, and Z 2 have the abovementioned meaning, b represents the integers 0 or 1, K is a chelating agent residue of the general formula II A or II B *0 0 (2HS-(H2n (Tj A) 0 R 3 -S4 *R 5 -S (CH 2 C -NH 0 wherein R 2 R 3 and RS are, independent of each other, a hydrogen atom, a (C 1 6 alkyl)CO, (C,5- 6 aryl)CO, or (C 7 arylalkyl)CO residue, optionally substituted with a hydroxyl group, a C 1 4 alkoxy, a carboxyl, or a sulphonic acid group, and wD93aKNA.DOC R 4 represents a residue of either formula II C or II D -CH 2 -CH- I I C C=o I 0. I wherein the carbon atoms labelled with an asterisk are bound to the imino groups of formula II B, and wherein n' is an integer 1 or 2, i is any integer from 2 to 6, and 15 TT represents a- and/or p-amino acids linked together in the usual way through amide bonds; e Se as well as chelating agent residues derived from dithiosemicarbazone derivatives of formula II E RT HN-N N-NH R 6 IIE) N=S =N SH HS ID938mA.DOC wherein R 6 is a hydrogen atom or a C.g 6 alkyl group; as well as chelating agent residues derived from bis(aminothiol) derivatives of formula II F I I F) r S S S *OS S S 5 0 9*e* 9 S 5 S wherein R 7 to R 18 represent, independent of each other, a hydrogen atom, a C1- 10 alkyl chain and/or an L- linkage, while o, p, r are either integers 1 or 2; as well as chelating agent residues derived from propylene aminoxime derivatives of formula II G /'CH2), R21 NH NH R2 R0 23 I IG wherein R 19 to R 24 represent a hydrogen atom or a C_. 4 alkyl residue, either identical or different, and independent of each other, is either integer 2 or 3; ID93SENA.DOC as well as chelating agent residues derived from diamido-dimercapto derivatives of formula II H Y 1 Y2 NH X, HN Y3 A, A 3 A 2 S S A 4 5 T T wherein X is a bond, a methylene group, or a CHY 4 :group, with one of groups Y 1 Y 2 Y 3 or Y4 representing an L-linkage and the others hydrogen atoms or optionally an oxygen atom, T is a hydrogen atom, an alkaline metal ion, a C1-6 acyl group, a benzoyl group, a S 15 hydroxyacetyl group, an acetamido methyl group, a p-methoxy benzyl group, an ethoxy ethyl group, or any other suitable SH protective group, AI, A 2 A 3 and A 4 are hydrogen atoms or C._ 6 alkyl groups, either identical or different, and independent of each other; as ill as chelating agent residues derived from diamido-dimercapto derivatives of formula II J ID938tA.DOC A A Y I Y NsZ Z N, wherein R 27 represents a hydrogen atom, or a C. 6 alkyl 5 residue optionally substituted with one or two hydroxyl groups, R 25 and R 26 are a hydrogen atom each, or an oxygen atom together A is a hydroxyl or mercapto group, Y is a hydrogen atom, a carboxy or sulphonyl residue, and Z is a carbon atom or a nitrogen atom; as well as chelating agent residues derived from amino polycarboxylic acids of either formula II K or II L CH 2 CO- CHX CH 2 X CH 2 X I I I N (CH 2 -CH 2 CH-- CH 2 (N-CH 2 N I I K I I CH 2 X' CH 2 X ID93 EA.DOC U R, CH 2 X I I I N CH (CH 2 N (CH 2 )a CH 2 IIL N ((CH 2 )a-)q CH 2 X CH 2 X wherein n and m are either integer 0, 1, 2, 3, or-4, 5 with n plus m not exceeding 4, a is an integer 2, 3, 4, or k is an integer 1, 2, 3, 4, or 1 is an integer 0, 1, 2, 3, 4, or 5, and q is an integer 0, 1, or 2, U represents a hydrogen atom, or a C 1 -6 alkyl group optionally substituted with one or several hydroxy groups and containing an Linkage, S. each residue X represents hydrogen atoms, COOH groups, ester groups, or amide groups with 1 to 6 carbon atoms in the alkyl residue, independent of each other, RI is a binding to L or a hydrogen atom; as well as chelating agent residues of formula II M, Cp(aa)Cp- (II M) wherein Cp is a protected cysteine and (aa) one of the naturally occurring amino acids; ID938 NA.DOC as well as cysteine-rich amino acid sequences of the metalloid thionines -ser-cys-thr-cys-thr-ser-ser-cys-ala-, -ala-cys-lys-ala-cys-lys-cys-, -gly-cys-ser-lys-cys-ala-gln-gly-cys-val-, -cys-lys-gly-ala-ala-asp-lys-cys-thr-cys-cys- ala- and analogous sequences in which serine has been replaced by threonine, glycine, or alanine; as well as endotheline derivatives, endotheline partial sequences, endotheline analogues or endotheline antagonists that are labelled using S 15 radioactive iodine isotopes. 2. Compounds according to Claim 1, characterized in that the metal ion M is an ion of an isotope of Tc, Re, In, Cr, Mn, Fe, Co, Ni, Cu, Pr, Nd, Sa, Y, Gd, Tb, Dy, Ho, Er, and La. 3. Compounds according to at least one of Claims 1 to 2, 25 characterized in that the endothelines E comprise the following sequence of amino acids cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr- phe-cys-his-leu-asp-ile-ile-trp or parts thereof. 4. Compounds according to at least one of Claims 1 to 2, characterized in that the parts of endothelines E comprise the amino acid sequence -his-leu-asp-ile-ile-trp-. ID938EBA.DOC Compounds according to at least one of Claims 1. to 2, characterized in that the endotheline analogues E comprise one of the following sequences of amino acids: -cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-va.-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- phe-cys-his-leu-asp-ile-ile-trp-; -cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr- phe-cys-his-leu-asp-ile-ile-trp-; 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-tnet-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-; or parts thereof. 6. Compounds according to at least one of Claims 2. to 2, characterized in that the endotheline antagonists E Ifl938A.flOC 79 comprise one of the following sequences of amino acids: -D trp-D asp-pro-D val-leu- or -D glu-ala-allo D ile-leu-D trp- 7. Compounds according to at least one of Claims 1 to 6, characterized in that the alkylene group representing L is unbranched, branched, cyclic, aliphatic, aromatic, or arylaliphatic. 8. Compounds according to at least one of Claims 1 to 6, characterized in that 15 L represents Z 1 -R-Z 2 wherein Z 1 and Z 2 are, independent of each other, a group, and R is an unbranched mono- to decamethylene group S or a residue of formula a (CH 2 Z 2 -(CH2)I (B) wherein s equals 1 and t equals 0, ring B is phenylene, and Z 1 and Z 2 are, independent of each other, a -NH-, group. 9. Compounds according to at least one of Claims 1 to 8, characterized in that the complexing agent residue K comprises ID93JSW.DOC 15 a 4-carboxyethylphenylglyoxal-bis- (N-rnethylthio- semicarbazone) -N-hydroxysuccinimide ester residue, a 6-(4'-isothiocyanatobenzyl) -3,3,9,9-tetramethyl-4,8- diazaundecane-2, lO-dion-dioxime residue, a 2-rnethyl-2- (4-isothiocyanatobenzyl) -propylene- bis-salicylidenamine residue, a 2-methyl-2-(4--isothiocyanatobenzyl) -propylene- bis- (suipho) salicylidenamine residue, a N,N'-bis(2-mercaptopyridyl)rnethyl] -2-rnethyl-2- (4- isothiocyanatobenzyl) -1,3-propanediamine residue, an S-benzoylthioacetylglycylglycyl glycine residue, a N,N' -bis (benzoylthioacetyl) 3-diaminopropionic acid residue a N,N' -bis (benzoylthioacetyl) -3,4-diaminobutyric acid residue, a N,N' -bis (benzoylthioacetyl) acid residue, a N,N' -1,2-ethylene diyl-bis- (2-iercapto-l-carboxy- ethylamine) residue, a cys (acm) -gly-cys (acm) -gly-gly- arg-gly- asp- ser residue, an ethylenediaminotetraacetic acid residue, a diethylenetriaminopentaacetic acid residue, ID93SRNA.DOC 81 a trans 2-cyclohexane diaminotetraacetic acid residue, a 1,4,7, 10-tetraazacyclododecanetetraacetic acid residue, a 1,4, 7-triazacyclononanetriacetic acid residue, a 1,4,8,11-tetraazatetradecane tetraacetic acid residue, a 1,5,9-triazacyclododecanetriacetic acid residue, a 1,4,7,1O-tetraazacyclododecanetriacetic acid residue, or a 3,6,9,15-tetraazabicyclo[9,3,15] -pentadeca- 1(15) ,11,13-trien-triacetic acid residue. ~:::*1Q.C'omplexes of compounds of the general formula (I) containing metal ions as defined in Claim 1: a) 99 mTc complex of S-benzoylthioacetyl-gly-gly-gly- gly-asp-his-leu-asp-ile-ile-trp, b) 99 mTc complex of N,N'-bis(S-benzoylthioacetyl)-3,4- diaminobutyryl-cys-ser-cys-ser-ser-leu-met-asp- lye- glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, c) 9 9 mTc complex of 3-[p-phenylglyoxal-di(N-methy'Athio semicarbazone)) -propionyl-cys-ser-cys-ser-ser-trp- leu-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp- ile-ile-trp, ZD93SBA.DQC d) 9 9 mTc complex of 3,6-diaza-1,8-dinercapto-2,7-bis- (carbonyl-gly-his-leu-asp-ile-ile-trp) octane, e) 9 9 mTc complex of {[(3,3,9,9-tetr:amethyl-4,8-diaza- undecyl 10O-dion-dioxime) 6-yljmethylphen-4' -yl} aminothiocarbonyl -cys -ser-ala-ser-ser-leu-met-asp- lys-glu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile- t rp, f) 99 'mTc complex of diamino-l-oxo-pentyl-cys-ser-cys-ser-ser-leu-met- asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile- ile -trp, 15 g) 9 9mTc complex of {[2,6-diaza-l,7-di(2-hydroxy- phenyl) -4-methyl-hept-4-yl]-methyl-phen-4' -yl} aminothiocarbonyl -cys-thr-cys-phe-thr-tyr-lys-asp- lys-glu-cys-val-tyr-tyr-cys--his-leu-asp-ile-ile- t rp, h) 9 9 mTc complex of {[2,6-diaza-l,7-di(2-hydroxy-5- *0*SSsulphophenyl) -4-methyl-hept-4-yl]-methylphen-4' -yl) aminothiocarbonyJl-ala-ser-ala--ser-ser-leu-met-asp- lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile- *trp, i) 99 mTc complex of N-[3,6,9-triaza-l-oxo-3,6,9,9- tetra- (hydroxycarbonylmethyl) -nonyl]-gly-his-leu- asp-ile-ile-trp, k) 11 1 1n complex of N-[3,6,9-triaza-1-oxo-3,6,9,9- tetra- (hydroxycarbonylnethyl) -nonyl]-gly-his-leu- asp-ile-ile-trp, ID938MMA.DOC 83 1) Gd(III) complex of N-(3,6,9-triaza-1-oxo-3,6,9,9- tetra- (hydroxycarbonylmethyl) -nonylj-gly-his-leu- asp-ile-ile-trp, sodium salt, m) Gd(III) complex of 1-{2-hydroxy-3-E4-(gly-his-leu- asp- ile- ile- trp-thiouridyl) phenoxyl propyll 1, 4,7,lO0-tetraaza-4,7,lO0-tris- (carboxylatomethyl) cyclododecane, n) 9 9 mTc complex of cys (acm) -gly-cys (acm) -cys-ser-cys- ser- ser- leu-net -asp- lys -glu- cys -val -tyr-phe -cys his-leu-asp-ile-ile-trp, o) 9 9 mTc complex of cyclo(trp-leu-val-pro-asp) cys(acm)-gly-cys(acm), p) 99 mTc complex of 4-mercaptobutyrimidyl-cys-ser-ala- ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys- :::*:his-leu-asp-ile-ile-trp, r 9 9 mTc complex of 3-thiopropionyl-gly-asp-his-leu- asp-ile-ile-trp, 99 mTc complex of 2- (acetylthio) succinyl-gly-asp- his-leu-asp-ile-ile-trp, t) 9 9 mTc complex of cys- ser-cys -ser-ser-leu-met -asp- lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile- trp. l1.Compounds according to Claim 1, characterized in that the radioactive iodine isotopes are 1311, 1251, and

1231. IID93Bma.DOc 84 12.Compounds of the general formula (I) E-L-(K)b (I) wherein E is a residue derived from endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists, L is a direct linkage or a Z 1 -R-Z 2 residue, wherin R is a unbranched, branched, saturated or 15 unsaturated C1- 20 -alkyl group, optionally interrupted by one or several oxygen and/or sulphur atoms and/or carbonyl, -NHCO-, -N(C_g 6 alkyl)CO-, -NH- and -N(Ci_6 alkyl) groups, and optionally substituted with hydroxy and/or epoxy groups; Z 1 and Z 2 are, independent of each other, an or group, or a residue of formula a S(CH 2 Z 2 Z, -(CH 2 s a wherein s and t are, independent of each other, the integers 0, 1, 2, or 3, ring B represents a phenyl or cyclohexyl group, and Z 1 and Z 2 have the abovementioned meaning, ID93BBA.DOC b represents the integers 0 or 1, K is a chelating agent residue of the general formula II A or II B o 0 R2-S(CH2W-C(TT~- CI I A) 0 S (CH 2 C-NH ~R 4 (I I B) R 5 S (CH 2 C -NH 0 wherein R 2 R 3 and RS are, independent of each other, a hydrogen atom, a (Cl- 6 alkyl) CO, (C 6 8 aryl) C0, Or (C 7 arylalkyl)CO residue, optionally substituted with a hydroxyl group, a C- alkoxy, a carboxyl, or a suiphonic acid group, and R 4 represents a residue of either formula II C or II D -CH 2 -CH- I I C) !fl93S11M.M~c I 86 -CH-(CH,) 2 (I" C=O wherein the carbon atoms labelled with an asterisk are bound to the imino groups of formula II B, and wherein n' is.an integer 1 or 2, i is any integer from 2 to 6, and TT represents a- and/or P-amino acids linked 10 together in the usual way through amide bonds; as well as chelating agent residues derived from 15 dithiosemicarbazone derivatives of formula II E /r( R HN N N-NH R 6 IE) N. N SH HS wherein R 6 is a hydrogen atom or a CI.- alkyl group; as well as chelating agent residues derived from bis(aminothiol) derivatives of formula II F ID938ENA.DOC 87 R /RU R Rw J SR NH NH R 1 R R 1 7 Ro R] (IIF) R 7 SH HS R, wherein R 7 to R 18 represent, independent.of each other, a hydrogen atom, a C1-10 alkyl chain and/or an L- linkage, while o, p, r are either integers 1 or 2; as well as chelating agent residues derived from 10 propylene aminoxime derivatives of formula II G CH2 L 4 NH NH ,R2 R20 23 Rg N N R 24 I I OH OH wherein R 19 to R 2 4 represent a hydrogen atom or a C,_4 alkyl residue, either identical or different, and independent of each other, m' is either integer 2 or 3; as well as chelating agent residues derived from diamido-dimercapto derivatives of formula II H ID938ENA.DOC 88 Yi Y2 NH X, HN Y3 S II H A, A 3 A 2 S S A 4 I I T T wherein X 1 is a bond, a methylene group, or a CHY 4 5 group, with one of groups YI, Y 2 Y 3 or Y4 *representing an L-linkage and the others ydrogen atoms or an oxygen atom, if any, T is a hydrogen atom, an alkaline metal ion, a C1-6 acyl group, a benzoyl group, a hydroxyacetyl group, an acetamido methyl group, a p-methoxy benzyl group, an ethoxy ethyl group, or any other suitable SH protective group, 15 A l A 2 A 3 and A 4 are hydrogen atoms or C.-6 alkyl groups,. either identical or different, and independent of each other; as well as chelating agent residues derived from diamido-dimercapto derivatives of formula II J R 27 R 2 R 2 NH NH 6 SA A Y I Y ID938rNA.DOC wherein R 2 7 represents a hydrogen atom, or a C-6. alkyl residue optionally substituted with one or two hydroxyl groups, R 25 and R 26 are a hydrogen atom each, or an oxygen atom together A is a hydroxyl or mercapto group, Y is a hydrogen atom, a carboxy or sulphonyl residue, and Z is a carbon atom or a nitrogen atom; eo as well as chelating agent residues derived from 15 amino polycarboxylic acids of either formula II K or II L CH 2 CO- CH 2 X CH 2 X CH 2 X N (CH 2 -CH2-N)- H CH 2 (N-CH 2 N I K) CH 2 X CH 2 X U R, CH 2 X N- (CH 2 )k CH (CH 1 N I I (CH 2 )o CH 2 IIL) N ((CH 2 0 o-)q CH 2 X CH 2 X wherein n and m are either integer 0, 1, 2, 3, or 4, with n plus m not exceeding 4, a is an integer 2, 3, 4, or ID938ENA.DOC k is an integer 1, 2, 3, 4, or 1 is an integer 0, 1, 2, 3, 4, or 5, and q is an integer 0, 1, or 2, U represents a hydrogen atom, or a CI_ 6 alkyl group optionally substituted with one or several hydroxy groups and containing an L-linkage, each residue X represents hydrogen atoms, COOH groups, ester groups, or amide groups with 1 to 6 carbon atoms in the alkyl residue, independent of each other, R 1 is an L-linkage or a hydrogen atom; *0 as well as chelating agent residues of formula II M, Cp(aa)Cp- (II M) wherein Cp is a protected cysteine and (aa) one of the 20 naturally occurring amino acids; as well as cysteine-rich amino acid sequences of the metalloid thionines -ser-cys-thr-cys-thr-ser-ser-cys-ala-, -ala-cys-lys-ala-cys-lys-cys-, -gly-cys-ser-lys-cys-ala-gln-gly-cys-val-, -cys-lys-gly-ala-ala-asp-lys-cys-thr-cys-cys- ala- and analogous sequences in which serine has been replaced by threonine, glycine, or alanine. 13.Compounds according to Claim 12, characterized in that the endothelines E include the following sequence of amino acids ID938EA.DOC 15 :25 cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val -tyr- phe-cys-his-leu-asp-ile-ile-trp or parts thereof. 14.Compounds according to Claim 12, characterized in that the parts of endothelines E comprise the amino acid sequence -his-leu-asp-ile-ile-trp-. 15.Compounds according to Claim 12, characterized in that the endotheline analogues comprise one of the following sequences of amino acids -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-th-r-tyr-lys-asp-Jlys-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- phe-cys-his-leu-asp-ile-ile-trp-; _-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-; ID938KNA.W~C -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-ys-glu-ala-vltyr- phe-ala-his-leu-asp-ile-ile-tp-; or parts thereof. 16.Compounds according to Claim 12, characterized in that the endotheline antagonists E comprs onIfh following sequences of amino acids: -D trp-D asp-pro-D val-leu, or *0 -D glu-ala--allo D ile-leu-D trp-. 17.Compounds according to at least one of Claims 12 to G 16, characterized in that the alkylene group representing L is unbranched, branched, cyclic, aliph~tic, aromatic, or arylaliphatic. 18.Compounds according to at least one of Claims 12 to 16, characterized in that L stands for Z 1 -R-Z 2 where in Z, and Z 2 are, independent of each 6ther, a -NH- group, and R is an unibranched mono-, to decamethylene group or for a residue of formula a ID93 8IUM.DOC (CH9t- Z 2 Z, -(CH 2 )s B a where in s equals 1 and t equals 0, ring B is phenylene, and Z, and Z 2 are, independent of each other, a -NH- -NiH-, group. 19.Cornpounds according to at least one of Claims 12 to 18, characterized in that the complexing agent residue K comprises a 4-tarboxyethylphenylglyoxal-bis- (N-methylthio- semicarbazone) -N-hy idroxysuccinimide ester residue, a 6-(4'-isothiocyanatobenzyl)-3,3,9,9-tetramethyl-4,8- diazaundecane-2 l-dion-dioxime residue, a 2-methyl-2- (4-isothiocyanatobenzyl) -propylene- ****bis-salicylidenamine residue, a 2-methyl-2- (4-isot -hiocyanatobenzyl) -propylene- bis- (suipho) salicylidenamine residue, a N,N'-bisII2-mercaptopyridyl)methyl] -2-methyl-2- (4- isothiocyanatobenzyl) -1,3-propanediamine residue, an S-benzoylthioacetylglycylglycyl glycine residue, a N,N' -bis (bmnzoylthioacetyl) -2 ,3-diaminopropionic acid residue a N,N' -bis (benzoylthioacetyl) -3,4-diaminobutyric acid XD938MM.DOC residue, a N,N' -bis (benzoylthioacetyl) acid residue, a N,N' -1,2-ethylene diyl-bis- (2-mercapto-1-carboxy- ethylamine) residue, a cys (acm) -gly-cys (acm) -giy-gly-arg-gly-asp-ser residue, an yeeimnttaaei cdrsde *an dethyleneiaminoetaacetic acid residue, a diethylenetriaminopnt acet acicesiduei a 147vetrans-, 2 cycloeaneintetraacetic acid residue, a 1,4 ,7,1O -tetraazacycloddecane tetraacetic acid residue, a 1,4,7-triazacyclononeanetriacetic acid residue, a 1,4,7, 1O-tetraazacyclododecanetriacetic acid residue, or a 3,6,9,15-tetraazabicyclo[9,3,15] -pentadeca.- 1 (15) ,11, 13-trien-triacetic acid residue. 1D93 BNA .DDC for preparing complexes of compounds of the general formula containing metal ions, characterized in that a) a radioactive metal ion in the form of its permetallate is reacted in a way known in the art with a compound of the general formula (I) E-L-(K)b (I) in the presence of a reducing agent and optionally an auxiliary ligand, or **e b) a suitable salt or oxide of a suitable paramagnetic 15 and/or radioactive cation is reacted in a way known in the art with a compound of the general formula (I) E-L-(K)b (I) o* or o000: :0..0 c) the disulphide bridges present in the endothelines, endotheline derivatives, partial endotheline sequences, endotheline analogues, or endotheline 25 antagonists are cleaved in a way known in the art, or that free thiol groups are produced using suitable methods such as the Trauts method which react with the radioactive metal ion in the form of a permetallate and in the presence of a reducing agent and optionally an auxiliary ligand, or d) radioactive iodine isotopes are introduced in the compound of the general formula II in a way known in the art using the chloramine-T method, or the ID938ENA.DOC lactoperoxidase method, or the Bolton-Hunter method, or the iodogen method, or another known method. 21.Method according to Claim 20a, characterized in that technetium-99m or Re are used in the form of pertechnetate or perrhenate. 22.Method according to Claim 20b, characterized in that 111 In is used as a radioactive cation. 9** 23.Method according to Claim 20b, characterized in that 15 Gd is used as a paramagnetic cation. 24.Method according to Claim 20c, characterized in that technetium-99m or Re are used in the form of 20 pertechnetate or perrhenate. according to Claim 20d, characterized in that 131I, 125I, or 123I is used as a radioactive iodine isotope. 26.Method for preparing a compound of the general formula characterized in that an endotheline, endotheline derivative, endotheline partial sequence, endotheline analogue, or endotheline antagonist is reacted with a compound of the formula (III) (K)b-L-H (III) wherein K, L, and b have the meaning given in Claim 1. ID938ENA.DOC 27.Method according to Claim 26, characterized in that E and L are linked through an ester, ether, thioether, thioester, amide, or thioamide linkage. 28.Diagnostic agent, characterized by the content of a compound according to Claims 1 to 11 and suitable excipients and carriers. oe* 29.Agent according to Claim 23, characterized in that the complex of the compound of the general formula (I) contains a radioactive metal ion. 30.Agent according to at least one of Claims 28 and 29, characterized in that the radioactive metal ion is Tc, Re, In, or I. 31.Agent according to Claim 28, characterized in that the complex of the compound of the general formula (I) 25 contains a paramagnetic metal ion. 32.Agent according to Claim 31, characterized in that the paramagnetic metal ion is Gd, Tb, Dy, Ho, Er, or Fe. 33.Agent according to Claim 28, characterized in that the metal ion shows a sufficient absorption for X-rays. 34.Cold kit for the preparation of radiopharmaceuticals comprising of a compound of the general formula (I) ID98 EOA.DOC according to one of Claims 12 to 19, a reducing agent and optionally an auxiliary ligand, present either in dry or dissolved form, directions for reacting the described compound with technetium-99m or Re in the form of a pertechnetate or perrhenate solution. for imaging pathological vascular changes, characterized in that an agent according to one of the Claims 28 to 33 is used as a contrast medium. S. 36.Method according to Claim 35, characterized in that the contrast medium is used for radiodiagnostics. 4 37.Cold kit containing endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists 20 bonded to a peptide, a derivative, or a chelating agent that are capable of binding metal ions, and optionally metal ions bonded to the latter. 25 38.Cold kit according to Claim 37, characterized in that the bound metal ion is not radioactive and can be easily replaced by a radioactive isotope. 39.Hot kit containing endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists bonded to a peptide, a derivative, or a chelating agent, and a radioactive iodine atom bonded to the latter. ID93BENA.DOC 99 of the cold kit according to Claim 37 whereby tne cold kit comprises a closeable vessel containing the predetermined quantity of endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists bonded to a peptide, a derivative, or a chelating agent capable of binding metal atoms, and containing a quantity of a reducing agent sufficient to label the compound with 99mTc in order to produce a radiopharmaceutical formulation. 41. Compounds of the formula methods for their manufacture or pharmaceutical compositions or methods of treatment involving/containing them, substantially as hereinbefore described with reference to the Examples. DATED this llth day of January 1994, INSTITUT FUR DIAGNOSTIKFORSCHUNG GMBH *98 AN DEER FREIEN UNIVERSITAT BERLIN By Its Patent Attorney DAVIES COLLISON CAVE ID938fNA.DOC ABSTRACT The present invention relates to metal complexes and me- tal complex conjugates of endothelines, endotheline de- rivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists, iodinated endo- theline derivatives, endotheline partial sequences, endo- theline analogues, or endotheline antagonists, and means containing the said compounds, their preparation as .0 diagnostic agents, as well as methods for the manufactu- ring of the said compounds and agents. 1 In vivo applicable metal complexes and metal complex con- jugates of endothelines, endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists as well as iodinated endotheline derivatives, endotheline partial sequences, endotheline analogues, or endotheline antagonists are preferably used for the non-invasive imaging of vascular diseases. e* ID93BIM.DOC