AGENTS FOR THE DIAGNOSIS OF VASCULAR DISEASES

AGENTS DE DIAGNOSTIC DES MALADIES VASCULAIRES

English Abstract

ABSTRACT OF THE DISCLOSURE
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 such compounds, their preparation as
diagnostic agents, as well as processes for the manufactu-
ring of such compounds and agents.
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.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
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
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 Z1-R-Z2 residue,
wherein
R is a unbranched, branched, saturated or
unsaturated C1-20-alkyl group, optionally
interrupted by one or several oxygen and/or
sulphur atoms and/or carbonyl, -NHCO-, -N(C1-6
alkyl)CO-, -NH- and -N(C1-6 alkyl) groups, and
optionally substituted with hydroxy and/or epoxy
groups;
Z1 and Z2 are, independent of each other, an
-O-, -S-, -(C=O)O-, -NH-(C=S)NH-, -(C=O)-,
-(C=S)O-, -NH-, -NH-(C=O)-, or -NH-(C=S)- group,
or a residue of formula .alpha.

<IMG> (.alpha.)
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 Z1 and Z2 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
<IMG> (II A)
<IMG> (II B)
wherein
R2, R3, and R5 are, independent of each other, a
hydrogen atom, a (C1-6 alkyl)CO, (C6-8 aryl)CO,
or (C7-9 arylalkyl)CO residue, optionally
substituted with a hydroxyl group, a C1-4
alkoxy, a carboxyl, or a sulphonic acid group,
and

R4 represents a residue of either formula II C
or II D
<IMG> (II C)
<IMG> (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
TT represents .alpha.- and/or .beta.-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
<IMG> (II E)

wherein
R6 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
<IMG> (II F)
wherein
R7 to R18 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
<IMG> (II G)
wherein
R19 to R24 represent a hydrogen atom or a C1-4
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
<IMG> (II H)
wherein
X1 is a bond, a methylene group, or a CHY4
group, with one of groups Y1, Y2, Y3 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
hydroxyacetyl group, an acetamido methyl group,
a p-methoxy benzyl group, an ethoxy ethyl group,
or any other suitable SH protective group,
A1, A2, A3 and A4 are hydrogen atoms or C1-6
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

<IMG> (II J)
wherein
R27 represents a hydrogen atom, or a C1-6 alkyl
residue optionally substituted with one or two
hydroxyl groups,
R25 and R26 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
<IMG> (II K)

<IMG> (II L)
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 5
k is an integer 1, 2, 3, 4, or 5,
l 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 C1-6 alkyl
group optionally substituted with one or several
hydroxy groups and containing an 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,
R1 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;

as well as cysteine-rich amino acid sequences of
the metalloid thionines
<IMG> ,
<IMG> ,
<IMG> ,
<IMG>
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
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, or La.
3. Compounds according to Claims 1 or 2,
characterized in that the endothelines E comprise the
following sequence of amino acids
<IMG>
or parts thereof.
4. Compounds according to Claims 1 or 2,
characterized in that the parts of endothelines E
comprise the amino acid sequence
-his-leu-asp-ile-ile-trp-.

5. Compounds according to Claims 1 or 2,
characterized in that the endotheline analogues
comprise one of the following sequences of amino
acids:
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;
or parts thereof.
6. Compounds according to Claims 1 or 2,
characterized in that the endotheline antagonists E

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 Claims 1 or 2,
characterized in that the alkylene group representing
L is unbranched, branched, cyclic, aliphatic,
aromatic, or arylaliphatic.
8. Compounds according to Claims 1 or 2,
characterized in that
L represents Z1-R-Z2,
wherein
Z1 and Z2 are, independent of each other, a
-(C=O)O-, -(C=O)-, -NH-, -NH-(C=O)- group, and
R is an unbranched mono- to decamethylene group
or a residue of formula .alpha.
<IMG> (.alpha.)
wherein
s equals 1 and t equals 0, ring B is phenylene, and
Z1 and Z2 are, independent of each other, a
-NH-(C=S)-, -NH-(C=S)NH-, -(C=O)O-, -(C=O)-, -NH-,
-NH-(C=O)- group.
9. Compounds according to Claims 1 or 2,
characterized in that the complexing agent residue K
comprises

11
a 4-carboxyethylphenylglyoxal-bis-(N-methylthio-
semicarbazone)-N-hydroxysuccinimide ester residue,
a 6-(4'-isothiocyanatobenzyl)-3,3,9,9-tetramethyl-4,8-
diazaundecane-2,10-dion-dioxime residue,
a 2-methyl-2-(4-isothiocyanatobenzyl)-N,N'-propylene-
bis-salicylidenamine residue,
a 2-methyl-2-(4-isothiocyanatobenzyl)-N,N'-propylene-
bis-[5-(sulpho)salicylidenamine residue,
a N,N'-bis[2-mercaptopyridyl)methyl]-2-methyl-2-(4-
isothiocyanatobenzyl)-1,3-propanediamine residue,
an S-benzoylthioacetylglycylglycyl glycine residue,
a N,N'-bis(benzoylthioacetyl)-2,3-diaminopropionic
acid residue
a N,N'-bis(benzoylthioacetyl)-3,4-diaminobutyric acid
residue,
a N,N'-bis(benzoylthioacetyl)-4,5-diaminopentanoic
acid residue,
a N,N'-1,2-ethylene diyl-bis-(2-mercapto-1-carboxy-
ethylamine) residue,
a cys(acm)-gly-cys(acm)-gly-gly-arg-gly-asp-ser
residue,
an ethylenediaminotetraacetic acid residue,
a diethylenetriaminopentaacetic acid residue,

12
a trans-1,2-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 1,4,7,10-tetraazacyclododecanetriacetic acid
residue, or
a 3,6,9,15-tetraazabicyclo[9,3,15]-pentadeca-
1(15),11,13-trien-triacetic acid residue.
10.Complexes of compounds of the general formula (I)
containing metal ions as defined in Claim 1:
a) 99mTc complex of S- benzoylthioacetyl-gly-gly-gly-
gly-asp-his-leu-asp-ile-ile-trp,
b) 99mTc complex of N,N'-bis(S-benzoylthioacetyl)-3,4-
diaminobutyryl-cys-ser-cys-ser-ser-leu-met-asp-lys-
glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp,
c) 99mTc complex of 3-[p-phenylglyoxal-di(N-methylthio
semicarbazone)]-propionyl-cys-ser-cys-ser-ser-trp-
leu-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-
ile-ile-trp,

13
d) 99mTc complex of 3,6-diaza-1,8-dimercapto-2,7-bis-
(carbonyl-gly-his-leu-asp-ile-ile-trp) octane,
e) 99mTc complex of {[(3,3,9,9-tetramethyl-4,8-diaza-
undecyl-2,10-dion-dioxime)-6-yl]methylphen-4'-yl}
aminothiocarbonyl-cys-ser-ala-ser-ser-leu-met-asp-
lys-glu-ala-val-tyr-phe -cys-his-leu-asp-ile-ile-
trp,
f) 99mTc complex of N,N'-bis(S-benzoylthioacetyl)-4,5-
diamino-1-oxo-pentyl-cys-ser-cys-ser-ser-leu-met-
asp-lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-
ile-trp,
g) 99mTc complex of {[2,6-diaza-1,7-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-
trp,
h) 99mTc complex of {[2,6-diaza-1,7-di(2-hydroxy-5-
sulphophenyl)-4-methyl-hept-4-yl]-methylphen-4'-yl}
aminothiocarbonyl-ala-ser-ala-ser-ser-leu-met-asp-
lys-glu-ala-val-tyr -phe-ala-his-leu-asp-ile-ile-
trp,
i) 99mTc complex of N-[3,6,9-triaza-1-oxo-3,6,9,9-
tetra-(hydroxycarbonylmethyl)-nonyl]-gly-his-leu-
asp-ile-ile-trp,
k) 111In complex of N-[3,6,9-triaza-1-oxo-3,6,9,9-
tetra-(hydroxycarbonylmethyl)-nonyl]-gly-his-leu-
asp-ile-ile-trp,

14
l) Gd(III) complex of N-[3,6,9-triaza-1-oxo-3,5,9,9-
tetra-(hydroxycarbonylmethyl)-nonyl]-gly-his-leu-
asp-ile-ile-trp, sodium salt,
m) Gd(III) complex of 1-{2-hydroxy-3-[4-(gly-his-leu-
asp-ile-ile-trp-thiouridyl)phenoxy]propyl}
1,4,7,10-tetraaza-4,7,10-tris-(carboxylatomethyl)-
cyclododecane,
n) 99mTc complex of cys(acm)-gly-cys(acm)-cys-ser-cys-
ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-
his-leu-asp-ile-ile-trp,
o) 99mTc complex of cyclo(trp-leu-val-pro-asp)-
cys(acm)-gly-cys(acm),
p) 99mTc 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) 99mTc complex of 3-thiopropionyl-gly-asp-his-leu-
asp-ile-ile-trp,
s) 99mTc complex of 2-(acetylthio)succinyl-gly-asp-
his-leu-asp-ile-ile-trp, or
t) 99mTc complex of cys-ser-cys-ser-ser-leu-met-asp-
lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-
trp.
11.Compounds according to Claim 1, characterized in that
the radioactive iodine isotopes are 131I, 125I, and
123I,

12.Compounds of the general formula (I)
E-L-(R)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 Z1-R-Z2 residue,
wherin
R is a unbranched, branched, saturated or
unsaturated C1-20-alkyl group, optionally
interrupted by one or several oxygen and/or
sulphur atoms and/or carbonyl, -NHCO-, -N(C1-6
alkyl)CO-, -NH- and -N(C1-6 alkyl) groups, and
optionally substituted with hydroxy and/or epoxy
groups;
Z1 and Z2 are, independent of each other, an
-O-, -S-, -(C=O)O-, - NH-(C=S)NH-, -(C=O)-,
-(C=S)O-, -NH-, -NH-(C=O)-, or -NH-(C=S)- group,
or a residue of formula a
<IMG> (.alpha.)
wherein
s and t are, independent of each other, the
integers 0, l, 2, or 3,
ring B represents a phenyl or cyclohexyl group,
and Z1 and Z2 have the abovementioned meaning,

16
b represents the integers 0 or 1,
K is a chelating agent residue of the general
formula II A or II B
<IMG> (II A)
<IMG> (II B)
wherein
R2, R3, and R5 are, independent of each other, a
hydrogen atom, a (C1-6 alkyl)CO, (C6-8 aryl)CO,
or (C7-9 arylalkyl)CO residue, optionally
substituted with a hydroxyl group, a C1-4
alkoxy, a carboxyl, or a sulphonic acid group,
and
R4 represents a residue of either formula II C
or II D
<IMG> (II C)

17
<IMG> (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
TT represents .alpha.- and/or .beta.-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
<IMG> (II E)
wherein
R6 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

18
<IMG> (II F)
wherein
R7 to R18 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
<IMG> (II G)
wherein
R19 to R24 represent a hydrogen atom or a C1-4
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

19
<IMG> (II H)
wherein
X1 is a bond, a methylene group, or a CHY4
group, with one of groups Y1, Y2, Y3 or Y4
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
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,
A1, A2, A3 and A4 are hydrogen atoms or C1-6
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
<IMG> (II J)

wherein
R27 represents a hydrogen atom, or a C1-6 alkyl
residue optionally substituted with one or two
hydroxyl groups,
R25 and R26 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
<IMG> (II K)
<IMG> (II L)
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 5

21
k is an integer 1, 2, 3, 4, or 5,
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 C1-6 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,
R1 is an L-linkage 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;
as well as cysteine-rich amino acid sequences of
the metalloid thionines
<IMG> ,
<IMG> ,
<IMG> ,
<IMG>
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

22
<IMG>
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
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;

23
<IMG> ;
<IMG> ;
or parts thereof.
16.Compounds according to Claim 12, characterized in that
the endotheline antagonists E 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-.
17.Compounds according to Claim 12,
characterized in that the alkylene group
representing L is unbranched, branched, cyclic,
aliphatic, aromatic, or arylaliphatic.
18.Compounds according to Claim 12,
characterized in that
L stands for Z1-R-Z2,
wherein
Z1 and Z2 are, independent of each other, a
-(C=O)O-, -(C=O)-, -NH-, -NH-(C=O)- group, and
R is an unbranched mono- to decamethylene group
or for a residue of formula .alpha.

24
<IMG> (.alpha.)
wherein
s equals 1 and t equals 0, ring B is phenylene, and
Z1 and Z2 are, independent of each other, a
-NH-(C=S)-, -NH-(C=S)NH-, -(C=O)O-, -(C=O)-, -NH-,
-NH-(C=O)- group.
19.Compounds according to Claim 12,
characterized in that the complexing agent residue
K comprises
a 4-carboxyethylphenylglyoxal-bis-(N-methylthio-
semicarbazone)-N-hydroxysuccinimide ester residue,
a 6-(4'-isothiocyanatobenzyl)-3,3,9,9-tetramethy-4,8-
diazaundecane-2,10-dion-dioxime residue,
a 2-methyl-2-(4-isothiocyanatobenzyl)-N,N'-propylene-
bis-salicylidenamine residue,
a 2-methyl-2-(4-isothiocyanatobenzyl)-N,N'-propylene-
bis-[5-(sulpho)salicylidenamine residue,
a N,N'-bis[2-mercaptopyridyl)methyl]-2-methyl-2-(4-
isothiocyanatobenzyl)-1,3-propanediamine residue,
an S-benzoylthioacetylglycylglycyl glycine residue,
a N,N'-bis(benzoylthioacetyl)-2,3-diaminopropionic
acid residue
a N,N'-bis(benzoylthioacetyl)-3,4-diaminobutyric acid

residue,
a N,N'-bis(benzoylthioacetyl)-4,5-diaminopentanoic
acid residue,
a N,N'-1,2-ethylene diyl-bis-(2-mercapto-1-carboxy-
ethylamine) residue,
a cys(acm)-gly-cys(acm)-gly-gly-arg-gly-asp-ser
residue,
an ethylenediaminotetraacetic acid residue,
a diethylenetriaminopentaacetic acid residue,
a trans-1,2-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 1,4,7,10-tetraazacyclododecanetriacetic acid
residue, or
a 3,6,9,15-tetraazabicyclo[9,3,15]-pentadeca-
1(15),11,13-trien-triacetic acid residue.

26
20 Process for preparing complexes of compounds of the
general formula (I) 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
b) a suitable salt or oxide of a suitable paramagnetic
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)
or
c) the disulphide bridges present in the endothelines,
endotheline derivatives, partial endotheline
sequences, endotheline analogues, or endotheline
antagonists are cleaved in a way known in the art, or
that free thiol groups are produced using suitable
processes e.g. the Trauts process 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 process, or the

27
lactoperoxidase process, or the Bolton-Hunter process,
or the iodogen process, or another known process.
21.Process according to Claim 20a, characterized in that
technetium-99m or Re are used in the form of
pertechnetate or perrhenate.
22 Process according to Claim 20b, characterized in that
111In is used as a radioactive cation.
23.Process according to Claim 20b, characterized in that
Gd is used as a paramagnetic cation.
24 Process according to Claim 20c, characterized in that
technetium-99m or Re are used in the form of
pertechnetate or perrhenate.
25 Process according to Claim 20d, characterized in that
131I, 125I, or 123I is used as a radioactive iodine
isotope.
26 Process for preparing a compound of the general formula
(I), 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.

23
27.Process 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.
29.Agent according to Claim 28, characterized in that the
complex of the compound of the general formula (I)
contains a radioactive metal ion.
30.Agent according to Claims 28 or 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)
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)

29
according to Claim 12
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.
35.Method for imaging pathological vascular changes,
characterized in that an agent according to
Claim 28 is used as a contrast medium.
36.Method according to Claim 35, characterized in that
the contrast medium is used for radiodiagnostics.
37.Cold kit containing endothelines, endotheline
derivatives, endotheline partial sequences,
endotheline analogues, or endotheline antagonists
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.
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.

40.Use of the cold kit according to Claim 37 whereby the
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.

Description

21~324~
This lnvention 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, and compositions that contain
these compounds, their use as agents for diagnosis, and
the processes for preparing these compounds and
compositions. Metal complexes, metal complex conjugates
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 that are applicable
in vivo are used to yield images of vascular diseases,
preferably in a non-invasive way.
Atherosclerosis is a chronic, progressive disease of the
blood vessels which has as yet been clinically
diagnosable only at an advanced stage. Atherosclerotic
vascular alterations are conventionally depicted by means
of arteriography. A contrast medium is applied to the
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 e.g. pains, perforations of
arteries, arrhythmia, cardiac or cerebral infarcts which
in unfavourite circumstances may result in the patient's
death.
ID99~N .DOC

2 2~132 !1~
Furthermore, method~ baeed on the u~e of ul~ra~onic wave~
and MR eomography are u8ed to diag~oae athero6clerosi~
All methodg currently a~plied hav~ the great di3advantage
that thoy detece athe~o~clerotic ~a~cular changes ~rom
the dimini~hed ~lood flow or ~ignificant alteration~ of
th~ areery wall i~ the~e ~ection~ and are thu~ only
capable of de~ecting advanced ~tages of atherogene~
Early detection of athero~clerosiff would be of great
importance ~or monitoring the therapeutic e~fect of
dlet~, calcium antagoni~t~, lipid and hypertenaion
depre~ant~, for ~o~itoring re~tenosi~ after angioplastic
surgery, for diagnosing coronary heart disea~e6, and fo~
detecting thrombotic deposi~s in the ve~els.
Non-invaeive methods o~ diagno~ing athero~clero~i~ ha~e
b~en de w ribed before. Thu~, antibodies la~elled with
radlol~otopes, or labelled low-den~ity lipoprotein~
(LDL), wQre introduced that bond to atherosclerotlc wall ~
~ectione (Leee et al. 1983, J. Nucl. Med. 24, 154-156; ~;
Kal$man et al. 1385, Circulation, 72, 300; Vi~golini et
al. 1991, Eur. J. ~ucl. Med., 18, 944-947). Thee~ :
method~, however, have ma~o~ di~advantage~ ~.q, the
antiyenic effect o~ the antibodieo on the ~y~tem and the
long period of time (~e~eral day~) requlred to i~ola~e,
refine, and label the LD~ obtai~ed ~rom the patient~
blood. Above all, these big moleculcs have a long half-
li~e in the blood, which togcther with high background
radlation make~ it difficult, if not impo~ le, to
locate atherooclerotlc leeion~.
S~h et al. ~1990, Proc. Natl. Acad. Scl., B7, 1s36-l4so)
ha~e aynt-he~ized partlal ~equencee of the LDL protein
moiety (apo-B-~00) that ~tlll ~ond to the athero~clerotic
plaque~ but have a con~iderably ~ho~ter half-llf~ ~n ~he
~ '
., . - ~ .~ .. : . ............. .. : .
i~

~~` 3 2~ ~32~i`
blood and a~ improved e~g~a~-~oi~e ratio. A euccesa~ul in
vlvo diagnoeis of atheroBclero~is, however. could no~ be
e3tabli~hed with the~e apo-B-peptidee due to their lower
afflnity to th~ plaque and/or lower denslty of the
bondlng place~ ln ~he plaque.
It ie, therefore, an object of the invention to provide
new compounde and compoeitions that are ~uited for a new,
non-invaejive method of dlagnosie of, in particular, the
early, non-~tenotic ~ascular dl~eases. Thie object 1~
accompliehed by the metal complexe~ and metal complex
conjugate~ of endothelinen, endotheline derivativee,
endotheline partial seguences, endotheline analogue~ or
endoeheline antagoniet~, as well as iodinated endotheline
derlvativee, endotheline partial eeguence~, endotheline
analoguee, or endotheline antagonists according to the
lnvention.
Endothellnee aro phyeiologically_acti~e peptidee that
have both hormonic and neuroregulatory functions within
the organi~m (MacCumber et al. 1989, Proc. Natl. Acad.
Sci., a6, 72~5-7289; Yanagieawa et al. 19~9, Trends
Pharmacol. Sci., 10, 374-37~; LeMonier de Gouville et
al., 19~9, ~ife Sci., 45, 1499-1513; Yanagisawa et al.,
19~, Nature, 332, 411-415). Four dif~erent i50type9 have
ae yet been ~ound in man ~Inoue et al,, 1989, Proc. Natl.
Acad. Scl., 86, 2863-2867). Sndotheline 1 ig a
polypeptide coneietlng of the following eeque~ce of 21
ami~o acid~:
cye-eer-cye-eer-eor-leu-met-aep-lye-glu-cye-val-tyr-phe-
cy~-hie-leu-a3p-ile-ile-trp
~Yanagleawa et al., 19~, Nature, 332, 411-41S). An
inactive precureor of endotheline, the big endotheline,
ie ~ormed by the vaecular endothelium. Endotheline, which
I D~ I . DOI~

4 2 ~ 1 3 2 ~ 3
bond~ to ~pecific receptor~ of the smooth vascular
muacles, i~ formed aSter the endothel~ne-converting
enzyme (ECE) hae ~plit off a heptad~capeptide. There, it
reA~lts in a Ca~+-mediated contraction of the smooth
S mu~cle celle (Yanagisawa et al., l9B8, Nature, 332, 411-
415; Takuwa et al. 1991, Contrib~ Nephrol., 90, 99-104).
The proliferatlon of smooth mùscle celle in the ve~sel
wall due among other to growth factors (e.g. PDGF) i9 one
of the early irrever~ible cha~gee that occur during
at~erogenesi~ (Deemoulière and Gabbiani 1992,
Cere~rovasc. Di~., 2, 63-71; Ro~ 1986, N. Engl. J. Med.,
314, ~4~-5011. ~he~e proliferating cell~ alter their
morpholosy and their phy~iological function (Deemoulière
and Gabbiani 1992, Cerebrovasc. Dis., 2, 63-71). The in- ~`
vltro incubation of atheromatous human coronary arte~ies
with 125I-endotheline 1 ~howe~ that increa~ed bonding of
125I-endotheline 1 occur~ in the tunica media a~d the
vaea ~asora Bectione ~Da~hwood et al., 1991, J.
Cardio~asc. Pharmacol., 17, 45B-462). An increased uptake
of 125I-endotheline 1 in the deendothellated aortic
~ectione wa~ found when 125I-endothcllne 1 was applied to
rabbits who~e abdominal aorta had been deendotheliated
ucing a balloon catheter. This indicate6 a higher den~ity
of bonding places for endotheline 1 in the~e injured
vaecular s~ctione (Rurata et al. 1992, J. Nucl. Med., 33, ;~
a45). The etudie~ indicate that the proliferating emoot~
muacle cell~ keep expre~slng the endotheline recepeor. ~-
Endotheline 1 ha~ a ~trong vaeoconetricti~e effect on the
~mooth ~a~cular mu~culature (A. M. Doherty, 1992,
Medlclnal ~hemi~try, 35, 1493-150~ here~ore, only
relatively low concentration~ may be applied ~.~. to the
organi~m. Higher concentra~ions may be applied of -~
endoCheline partial ~equencee, endotheline analoguee or
endotheline anca~oniete, as they still bond to the
tD-~-~ll.~C

2~32~
endotheline receptor but do not cause such a distinct
contraction of the smooth muscle cells.
As endothelines are rapidly eliminated via the kidneys,
S 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
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 ~ ;
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
substancès 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
ID9338N.DDC

6 21~3~
antagonists, iodinated endotheline derivatives,
endotheline partial ~equences, 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
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 characterized, in that
E is a residue derived from endothelines,
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 antagonist~ carrying free thiol groups
and directly bindiny a metal ion;
L is a direct linkage or a Zl-R-Z2 residue,
wherein
R i~ a unbranched, branched, saturated or
unsaturated Cl 20-alkyl group, optionally
~D93 98N . DOC
~ : ~2 ~ ~ ?~

7 21 13 2!15
interrupted by one or several oxygen and/or
sulphur atoms and/or carbonyl, -NHCO-, -N(C1_6
alkyl)CO-, -NH- and -N(C1 6 alkyl) groups, and ~ ~9
optionally substituted with hydroxy and/or epoxy
groups;
Z1 and Z2 are, independent of each other, an
-O-, -S-, -(C=O)O-, -NH-(C=S)NH-, -(C=O)-,
-(C=S)O-, -NH-, -NH-(C=O)-, or -NH-(C=S)- group,
, ", ,~, "~ ~,
or a residue of formula a
(CH2)t - Z2
Z~--(CH2)5 {~ ( 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 Z1 and Z2 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
O O
11 11
R2 S- (CH2)n,- C- (TT);-C-- ( I I A )
.
:
ID931111N.DOC

21~32
O
R3 S (CH2)n~ C-NH
/R4 ( I I B ) ;~
R5 S ~ (CH2)n.--C ~ NH
O .
wherein
R2, R3, and R5 are, independent of each other, a
hydrogen atom, a (C1 6 alkyl)CO, (C6 8 aryl)CO, l-
or (C7 9 arylalkyl)CO residue, optionally . ~:
substituted with a hydroxyl group, a C1 4 ~ ::
alkoxy, a carboxyl, or a sulphonic acid group, ..
and -:
' ' ' " '~ ' ' '
R4 represents a residue of either formula II C .
or II D
~ : :.:
--CH2 CH- ( I I C ) ~ ::
C=O :.. ' ::""'
,,
--CH (CH2)2- ( 11 D )
C-O
.
wherein
. 20 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,
. . ~
~D93911N,DOt!

9 2 ~ ~ 3 2 ~
i is any integer from 2 to 6, and ~.
TT represents a- and/or ~-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 : :~
;:
R6 HN-N N-NH R6 ( I I E )
N =( )~ N
o SH HS
wherein :
R6 is a hydrogen atom or a Cl 6 alkyl group;
as well as chelating agent residues derived from
bis(aminothiol) derivatives of formula II F
Ru r R~
, Rn~R,~ ~
--Rlo ~NHNH~-- R6--
Rs ~ J ~ R~ (IIF )
R~ ~ ~ Rl7
R7 SH HS R~
wherein
R7 to Rl8 represent, independent of each other, a
: hydrogen atom, a Cl 10 alkyl chain and/or an L-
linkage, while o, p, r are either integers 1 or
2; ..
.: .
ID93111~N.DOC

2 1 L 3 2 ~
a~ well as chelating agent residues derived from
propylene aminoxime derivatives of formula II G
~CH2)m~
R21 NH NH R22
20--~ \~R2~
¦ (1I G ) ;
/ ~
R19 N N R24 ~ ;~
OH OH
wherein
Rlg to R24 represent a hydrogen atom or a Cl 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 ;
Y2 yNH X1 HNyY3
Az S S A4
T T
wherein
Xl is a bond, a methylene group, or a CHY4
group, with one of groups Yl, Y2, Y3 or Y4
representing an L-linkage and the others
hydrogen atoms or optionally an oxygen atom,
~D93BIIN.DOC

11 2~32~
T is a hydrogen atom, an alkaline metal ion, a ~:
Cl 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,
Al, A2, A3 and A4 are hydrogen atoms or Cl 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 ~/
R25----R2s
R26 'I H 1` R26
( ll J )
~ A A
wherein
R27 represents a hydrogen atom, or a Cl 6 alkyl
residue optionally substituted with one or two
hydroxyl grcups,
R25 and R26 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;
ID91 B~N . DOC

~~~ ` 12 2~ ~32~
as well as chelating agent residues derived from
amino polycarboxylic acids of either formula II K
or II L
CH2CO- CH2X CH2X CH2X ~,
N- (CH2-CH2-N)n--CH2--CH2--(N~CH2~CH2)m N ( I I K ) ~ ~
CH2X CH2X : ;:
~ `
U Rl CH2X
N- (CH2)k--CH--(CH2)1 N
(CH2)o
~cH2)a ~ ( I I L )
N ((CH2)0~l~J)q
CH2X CH2X
wherein
n and m are either integer 0, 1, 2, 3, or 4,
with n plus m not exceeding 4,
a i5 an integer 2, 3, 4, or 5
k is an integer 1, 2, 3, 4, or 5,
1 is an integer 0, 1, 2, 3, 4, or 5, and
IS q i8 an integer 0, 1, or 2,
: U represents a hydrogen atom, or a C1 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,
Rl is an L-linkage or a hydrogen atom;
SD9JJI~N.DOC

~ ` 13 21 L32~
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-,
-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
radioactive iodine isotopes.
2S
The metal ions contained in the complexes according to
the invention have properties that facilitate their
identification using physical methods. These -
properties are, for example, paramagnetism, radioactivity
including a-, ~-, and ~-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.
~D9398N.DOC

~` 21.L324~ :
14
Ions of isotopes of the following metals are particularly
preferred due to such 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 such ::
properties.
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, -
further contain residues E which derive from
lS 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 i9 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-phe-
::
cys-his-leu-asp-ile-ile-trp.
. .:
The hydrophobic region of endotheline 1, hisl6-trp2l 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.
~D9 3 3 liN . DOC

15 21~32'~ ~
Therefore, peptides that include the -his-leu-asp-ile-
ile-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 analogues.
The particularly preferred endotheline analogues
according to the invention comprise the following
sequences of amino acids or parts thereof:
,~ :
-cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-
cys-his-leu-asp-ile-ile-trp- (endotheline 2)
,.
-cy~-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr-tyr-
cys-his-leu-asp-ile-ile-trp- (endotheline 3)
,:,
-cys-ser-ala-eer-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-
cys-his-leu-asp-ile-ile-trp- (ala-endotheline)
'.' .
~ ,
-cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe~
I
cys-his-leu-asp-ile-ile-trp- (VIC)
-cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-aqn-phe-
cys-his-gln-asp-val-ile-trp- (sarafotoxin S 6a)
ID93011N.DOC
p`,.~ . !,,, `. ~,, ': ~ .' . . ~ ' , ~ ~ '

16 2t 132~
I I
-ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-
ala-his-leu-asp-ile-ile-trp- :
. .
-ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-
ala-his-leu-asp-ile-ile-trp-
-cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val-tyr-phe-
ala-his-leu-asp-ile-ile-trp-
Furthermore, residues E are preferred that derive from
endotheline antagonists.
Preferably used endotheline antagonists are cyclic ~.
pentapeptides e.g.
-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 se~uences,
endotheline analogues or endotheline antagonists.
: In preferred complexes according to the invention,
residue L is an unbranched, branched, cyclic, aliphatic, ~:
ID93al;N .DOC

~` 22 2~32-1~
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 99mTc and l86Re.
Other preferred compounds according to the invention are
endotheline derivatives, endotheline partial sequences, `
endotheline analogues or endotheline antagonists labelled
with radioactive iodine isotopes. The iodine
isotopes have radioactive properties, which makes them
detectable by physical methods. Particularly preferred
iodine isotopes are l3lI, l25I, or l23I.
,~
Another object of the present invention are new
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
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 Zi-R-Z2 residue,
wherein
ID91111N.WC

23 2~32~ -
R is a unbranched, branched, saturated or
unsaturated C1 2 0-alkyl group, optionally -
interrupted by one or several oxygen and/or sulphur
atoms and/or carbonyl, -NHCO-, -N(Cl 6 alkyl)CO-,
-NH- and -N(C1 6 alkyl) groups, and optionally
substituted with hydroxy and/or epoxy groups;
Z1 and 22 are, independent of each other, an -O-,
-S-, -(C=O)O-, -NH-(C=S)NH-, -(C=O)-, -(C=S)O-,
-NH-, -NH-(C=O)-, or -NH-(C=S)- group,
or a residue of formula a
~ (CH2)t Z2
Zl (CH2)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 : : .
Zl and Z2 have the abovementioned meaning,
b represents the integers 0 or 1,
,:, .. .
~ is a chelating agent residue of the general formula
II A or II B
O :~,
Il 11 :
R2 S-(CH2)n--C-(TT)I-C-- ( I I A ) ~
` ~'
:~,
ID93311N.DOC

:-~ 24 21~ 3~ 5
R
R3 S - (CH2)n'--C - NH
~R4 ( I I B )
R5--S - (cH2)n~ C - NH
O ,,
wherein
R2, R3, and R5 are, independent of each other, a -
hydrogen atom, a (Cl 6 alkyl)CO, (C6 8 aryl)CO, or
(C7_9 arylalkyl)CO residue, optionally substituted ~ -
with a hydroxyl group, a Cl 4 alkoxy, a carboxyl,
or a sulphonic acid group, and
R4 represents a residue of either formula II C or : ~:
II D .
,",.. .
--CH2- CH- ( I I C )
C=O ' :'
~ 15
--CH (CH2)2 ( 11 D )
C-O
wherein
the carbon atoms labelled with an asterisk (*) ..
are bound to the imino groups of formula II B,
and wherein
n' i8 an integer 1 or 2,
,~ i i9 any integer from 2 to 6, and
ID938BN.DOC

2 1 ~3 2L~
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
dithio semicarbazone derivatives of formula II E
` ,~
R~, HN-N N-NH R6 ( I I E )
N =( . )~ N ~ :
SH HS ~- :
wherein
R6 is a hydrogen atom or a Cl 6 alkyl group; ::~
as well as chelating agent residues derived from .
bis(aminothiol) derivatives of formula II F
: :::
R~ r Ru l
Rn~R~ ~ , ...
--R~ -I,NH NH~-- R~
R ~ I F ) ~ ~;
R, SH HS R,B
.: ,
wherein
R7 to R18 represent, independent of each other, a
hydrogen atom, a Cl 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
ID93aaN .DOC

-
26 21132~ -
~CH2)m~ ;
R20 ~/ NH NH~R23
l (ll G )
R19 N N R24
OH OH
wherein `. - ~:
Rlg to R24 represent a hydrogen atom or a Cl_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
Y~
Y2 yNH X1 HNyY3
A2 S S A4
T T
wherein
X1 is a bond, a methylene group, or a CHY4 group,
with one of groups Yl, Y2, Y3 or Y4 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 C1 6
acyl group, a benzoyl group, a hydroxyacetyl group,
an acetamido methyl group, a p-methoxy benzyl
ID9331~!1.DOC
,~ .

` 2~3~
: 27 ~:
group, an ethoxy ethyl group, or any other suitable
SH protective group,
. , .
Al, A2, A3 and A4 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 ~ .:
1 0
R27 / '. .'
R25 ~ R25 . , .~ . .
R26 ~ H N~ R26 ( J ) ~.
~ A A
wherein
R27 represents a hydrogen atom, or a Cl 6 alkyl
re~idue optionally substituted with one or two
hydroxyl groups,
R25 and R26 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
:
ID93311N.DO~ ~ ~
.

~ 28 2~324~ -
CH2CO- CH2X CH2X CH2X '~
N (CH2-CH2-N)n--CH2--CH2--(N~CH2~CH2)m--N ( I I K )
CH2X CH2X ','~
... ..
y R1 CH2X
N- (CH2)k CH--(CH2)1 IN ~ :
¦ (CH2)a
~CH2)a ~ ( I I L ) :
' J
~ ((CH2)o~~)q :
CH2X CH2X
wherein
n and m are either integer 0, 1, 2, 3, or 4, with n
plu8 m not exceeding 4, ~:~
a i8 an integer 2, 3, 4, or 5
k is an integer 1, 2, 3, 4, or 5,
l 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 Cl 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,
Rl i8 an L-linkage or a hydrogen atom;
: as well as chelating agent residues of formula II M,
Cp(aa)Cp- (II M)
wherein
,'~ , '
ID93 311N . DOC

29 2 1 1 3 2 ~
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-,
-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.
The residues E of the complexes according to the
invention of compounds of the general formula (I)
E-L-(K)b (I)
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-phe-
I
cys-his-leu-asp-ile-ile-trp.
The hydrophobic region of endotheline 1, hisl6-trp2l is
essential for bonding to the receptor (Kimura et al.,
1988, Biochem. Biophys. Re~. Comm., 156, 1182-1186).
Furthermore, residues are preferred that derive from
endotheline partial sequences.
~ :
~D939BN.DOC

; ~ 30 2~32~
Therefore, peptides that include the -his-leu-asp-ile-
ile-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 analogues.
The particularly preferred endotheline analogues
according to the invention comprise the following
sequences of amino acids or parts thereof:
. .,
-cys-ser-cys-ser-ser-trp-leu asp-lys-glu-cys-val-tyr-phe-
cys-his-leu-asp-ile-ile-trp- (endotheline 2)
-cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr-tyr-
cys-his-leu-asp-ile-ile-trp- (endotheline 3)
-cys-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-
I
cys-his-leu-asp-ile-ile-trp- (ala-endotheline)
-cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-
,~ ;
cys-his-leu-asp-ile-ile-trp- (VIC)
..~
I -- I
-cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn-phe-
cys-his-gln-asp-val-ile-trp- (sarafotoxin S 6a)
ID9338N.DOC

31 21 l3 24~ ~
: '
~.-
-ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-
ala-his-leu-asp-ile-ile-trp-
S
-ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-
ala-his-leu-asp-ile-ile-trp- ~ -
-cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val-tyr-phe-
ala-his-leu-asp-ile-ile-trp-
Furthermore, residues E are preferred that derive from ~-
endotheline antagonists. ~ ;~
Preferably used endotheline antagonists are cyclic
pentapeptides e.g.
.'::
-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)
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 Zl-R-Z2, ~`
wherein are:
Zl and Z2~ independent of each other, a -(C=O)O-,
-(C=O)-, -NH-, -NH-(C=O)- group, and
R is an unbranched mono- to decamethylene group
ID939~;N. DOC

32 2 ~ 1 3 2 ~ ~
or for a residue of formula a
~ (CH2)t - Z2
Z1 (CH2)s~ ( a )
wherein :
g equals 1 and t equals 0, and ring B is phenylene and
wherein are
Z1 and Z2l independent of each other, a -NH-(C=S)-,
-NH-(C=S)NH-, .-(C=O)O-, -(C=O)-, -NH-, -NH-(C=O)-
group.
Preferred complexing agent residues K of compoundsaccording to the invention of the general formula (I)
E-L-(K)b (I)
derive from 4-carboxyethylphenylglyoxal-bis-(N-methyl-
thio-semicarbazone)-N-hydroxysuccinimide ester, 6-(4'-
ieothiocyanatobenzyl)-3,3,9,9-tetramethyl-4,8-diaza-
undecane-2,10-dion-dioxime, 2-methyl-2-~4-isothiocyana- ~ :
tobenzyl)-N,N'-propylene-bis-~alicylidenamine, 2-methyl-
2-(4-isothiocyanatobenzyl)-N,N'-propylene-bie-[5- :~
(eulpho)ealicylidenamine, N,N'-bis[2-mercaptopyridyl)-
methyl]-2-methyl-2-(4-isothiocyanatobenzyl)-1,3-
propanediamine, S-benzoylthioacetylglycylglycyl glycine,
N,N'-bis(benzoylthioacetyl)-2,3-diaminopropionic acid,
N,N'-bie(benzoylthioacetyl)-3,4-diaminobutyric acid, ; .:
N,N'-bis(benzoylthioacetyl)-4,5-diaminopentanoic acid, : :~
N,N'-1,2-ethylene-diyl-bis-(2-mercapto-1-carboxy- ~.
ethylamine), Cy8 (Acm)GlyCys(Acm)GlyGlyArgGlyAspSer,
ethylenediaminotetraacetic acid, diethylenetriamino-
pentaacetic acid, trans-1,2-cyclohexane diaminotetra-
acetic acid, 1,4,7,10-tetraazacyclododecanetetraacetic ~ .
acid, 1,4,7-triazacyclononane~riacetic acid, 1,4,8,11- :
'~
,
~D91 lBN . DOC

33 2 1 ~ 3 2 ~
tetraazatetradecane tetraacetic acid, 1,5,9-triazacyclo-
dodecanetriacetic acid, 1,4,7,10-tetraazacyclododecane-
triacetic 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 Cl-C4 alkoxy groups. The following groups
shall be named as examples: methyl, ethyl, 2-hydroxy-
ethyl, 2-hydroxy-1-(hydroxymethyl)-ethyl, l-(hydroxy-
methyl)-ethyl, propyl, isopropenyl, 2-hydroxypropyl, 3-
hydroxypropyl, 2,3-dihydroxypropyl, butyl, isobutyl,
isobutenyl, 2-hydroxybutyl, 3-hydroxybutyl, 2-, 3-, and
4-hydroxy-2-methylbutyl, 2- and 3-hydroxyisobutyl, 2,3,4-
trihydroxybutyl, 1,2,4-trihydroxybutyl, pentyl, cyclo-
pentyl, 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,
piperidinyl, pyrazolidinyl, pyrazolinyl, piperazinyl,
morpholinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl
ring.
The complexes of compounds according to the invention of
the general formula (I) containing metal ions are
prepared using a process 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 (I)
~D93911N.DOC

~` 34 21~32~
in the known way, with a reducing agent and op~ionally an
auxiliary ligand, being present.
Preferred metal ions are 99mTc 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 processes 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 i981).
The complexes of compounds according to the invention of
the general formula (I) 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 (I).
Among the preferred radioactive metal ions is, for ; ;~
example 111In, 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 (e.g. nitrate,
acetate, carbonate, chloride, or sulphate) of the desired
metal is suspended or dissolved in polar solvents eOgO
water or hydrous alcohols, and reacted with the ap-
propriate amount of complexing ligands. If desired, -
existing acidic hydrogen atoms or acid groups may be
. ' ' '' .
ID93911N.WC

^~ 35 21 l3 24~
substituted by cations of inorganlc 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 e.g. 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.
The complexes according to the invention of compounds of
the general formula (I) 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 processes ~;
e.g. the Trauts process .
~:
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 litera-
ture, 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-mercapto-
ethanol according to the method described in EP 0 271 806
or in Nucl.Med.Biol. 1~, 607-609, 1992, and by labelling
them using technetium-99m in the presence of auxiliary
ID933~N.DOC

~` 36 2~321~
ligands e.g. pyrophosphate, gluconate, or glucohept-
onate and a reducing agent in a way known in the art, or
by reacting E' with suitable heterobifunctional cross-
linkers as, for example, N-succinimidyl-3-(2-pyridyldi-
thio) 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, e.g. imino thiolane, and by labelling
it then with technetium-99m or Re in a way known in the
.::
art. ~
- ::
The iodinated endotheline derivatives, endotheline
partial sequences, endotheline analogues or endotheline ;
antagonists can be prepared preferably with 131I, l25I, or
123I Using e.g. the chloramine-T process (Krohn
et al., 1977, BBA 490, 49), the lactoperoxidase method
(Kitamura et al., 1990, Biochem. Biophys. Res. Commun.
170, 497-503; Emori et al., 1990, Biochem. Biophys. Res.
Commun. 162, 217-223), the Bolton-Hunter method (Bolton
and Hunter, 1973, Biochem. J., 133, 529-539), the iodogen
method (Fraker and Speck, 1978, Biophys. Res. Commun., ~ ~
80, 849-857), or other known pr~cesses : ~ ::
.~
The compounds according to the invention of the general
formula (I) 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) - ;
(K)b-L-H (III)
wherein
ID99a8N.DOC

37 2~32~-~
E', L, K, and b have the meanings specified above.
The compounds of formula (I) are prepared using a process
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 E'. 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. ~ `
., , ~.
If the chelating agents K' are linked through a carboxyl
group in L, the latter is transformed according to the
processes known to a person skilled in the art, for
example, the carbodiimide process (Fieser, Reagents for
Organic Synthesis 10, 142), or by means of a mixed or
cyclic anhydride (Org. Prep. Proc. Int. 1975, l, 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 processes described by
Barany and Marrifield in The Peptides: Analysis,
~5 Synthesis, Biology. Academic Press, New York, 1980;
,-.,-=..0~

2 1 i 3 2 '~ ? J
38
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 process 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 S- :;
acylated derivatives of thioacidic acid or 3-
thiopropionic acid with di-, tri-, tetra-, penta-, or
hexapeptides. The respective carbonic acids are activated
by a process known to a person skilled in the art, for
example, according to the carbodiimide process (Fieser, -
Reagents for Organic Synthesis 10, 142), or using a mixed ;
or cyclic anhydride (Org. Prep. Proc. Int. 1975, l, 215)
or an activated ester (Adv. Org. Chem. Part B, 472). ;
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-
diaminopropionic acid or 1,3-diaminobutyric acid and
successive reaction of the resulting N,N'-dichloroacetyl
diaminocarbonic acids with alkali salts of thiocarbonic
acids. Another process 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 1,2-
diaminopropionic acid or 1,3-diaminobutyric acid.
'. :'
The respective carbonic acids are activated by a process
known to a person skilled in the art, for example,
ID9391;N.WC

2 1 ~ 3 2
`` 39
according to the carbodiimide process (Fieser, Reagents
for Organic Synthesis 10, 142), or using a mixed or
cyclic anhydride (Org. Prep. Proc. Int. 1975, 7, 215) or
an activated ester (Adv. Org. Chem. Part B, 472).
Chelating agents K' 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
principally prepared by reductive aminations of
substituted or non-substituted 1,2-diketo or 1,3-diketo
compounds with substituted, unsubstituted, protected or ~
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-
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
way known in the art.
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 1,3-
diaminoalkanoic acid (see EP 0248506) and successive
reaction of the resulting N,N'-dichloroacetyl
diaminocarbonic acids with alkali salts of thiocarbonic
acids. Another process for preparing chelating agents K'
of the general formula II H as known from literature ~see
US 4,897,255) consists in reacting suitable activated
(for example, NHS ester) and S-acylated derivatives of
ID99a8N.DOC
.

~ 4~ 2~ 324~
thioacidic acid or 3-thiopropionic acid with
diaminopropionic acid or 1,3-diaminobutyric ~cid. The
respective carbonic acids are activated by a process known
to a person skilled in the art, for example, according to
the carbodiimide process (Fieser, Reagents for Organic
Synthesis 10, 142), or using a mixed or cyclic anhydride
(Org. Prep. Proc. Int. 197S, 2, 215) or an activated
ester (Adv. Org. Chem. Part B, 472). The separation of
the SH protective groups is done after coupling to E'
according to the processes 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 o-
substituted with an additional carboxylic or sulphonic
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
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 process known to a person
skilled in the art, as, for example, described in the -
European Patent Application EP 0 512 661.
ID9~a3-.DOC

41 X1~32~
Chelating agents K~ of the general formula II M are
prepared according to the processes 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 process known to a
person skilled in the art, for example, according to the
carbodiimide process (Fieser, Reagents for Organic
Synthesis 10, 142), or using a mixed or cyclic anhydride
(Org. Prep. Proc. Int. 1975, l, 215) or 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 processes 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 in S-protected form. The
cysteine-rich amino acid sequences are separated from the
resin in an N-protected form. The free carboxyterminal
end is activated by a process 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, l, 215) or an activated ester (Adv. Org. Chem.
Part B, 472). The protective groups are removed after
coupling to E' according to the processes known to a
person skilled in the art from literature, for example,
using basic hydrolysis, reductive decomposition, etc.
.
~D93~ DOC

42 2 1 ~ 3 ~
(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 (I) 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
must be radioactive. This applies in particular to the
ions of elements having atomic numbers 27, 29, 30-32, 37-
39, 42-51, 62, 64, 70, 75, and 77. Preferred isotopes ;
are, for example, 99mTc, 186Re, and 111In. Endotheline
derivatives, endotheline partial sequences, endotheline
analogues or endotheline antagonists may also be
radiolabelled using iodine isotopes. 123I is, for
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
divalent and trivalent ions of elements having atomic
numbers 2~-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 radiod~iagnostics, the central ion must derive from an
ID933BN.DOC

i` 43 2 1 1 3 2 ~ ~ ~
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 æeries.
: .
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 e.g. 43Sc, 44Sc, 52Fe, 55Co, 68Ga and Cu
(Heiss, W. d.; Phelps M. E.; Positron Emission Tomography
of Brain, Springer Verlag Berlin, Heidelberg, New York
1983).
Another object of the present invention are processes for
preparing the agents for diagnosis according to the
invention.
The 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-
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 ( e.g. trometha-
mine), auxiliary ligand additives ( e.g. sodium citrate
or sodium tartrate), reductants ( e.g. tin(II)
chloride), or - if need be - electrolytes e.g. sodium
chloride, or - if need be - (one of) the auxiliary
agent(s) commonly used in galenics ( e.g. lactose,
methyl cellulose, mannitol) and/or tenside(s) (for
example, lecithlnes, Tween~, Myrj~). The composition of
~D919BN.DOC '~
,~; . : .

44 21~32~
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 102 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,
preferably 5 to 30 mCi, per application. Normally, a
solution of the agent according to the invention i5
applied as an intravenous, intra-arterial, or peritoneal -~
injection of 0.1 to S ml of the agent according to the
invention. Intravenous application is preferred. Details
of its handling and dosage are described, for example, in
"Radiotracer~ for Medical Applications", CRC press, Boca
Raton, Florida. The compounds according to the invention
are used for radiodiagnostic and radiotherapeutic
purposes in the form of their complexes with
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
ID9391~N.DOC

~ 45 21 1 3 2 '1 ~ -
or replaced at all, or not to a clinically relevant
extent.
~he pharmaceutic agents for NMR and radiodiagnostics
according to the invention are also prepared in a known
way by dissolving or suspending the complex compounds ac-
cording to the invention in a hydrous medium - while
optionally adding, the additives common in galenics - and
then optionally sterilizing the solution or suspension.
Suitable ad;litives are, for example, physiologically safe
buffers ( e.g. tromethamine), complexing agent
additives ( e.g. diethylene triamine pentaacetic acid),
or - if need be - electrolytes e.g. sodium chloride or
- if need be - antioxidants e.g. ascorbic acid.
The pharmaceutic agents according to the invention used
in NMR diagnostics contain preferably l~mol to 1 moI/l of
complex salt and are normally applied at dosages of 0.001
to 5 mmol/kg. They are meant for enteral and parenteral
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.
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.
'
ID9~98N.WC

-' 211 32~
46
Neutrallzation is done by inorsanic bases ( e.g~
hydroxides, carbonates or bicarbonates) of, for example,
sodium, potassium, lithium, magnesium, or calcium and/or
of organic bases e.g. prima~y, secondary, and tertiary
amines like ethanol amine, morpholine, glucamine, N-
methyl 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
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
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
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 ( e.g.
tromethamine), minor quantities of electrolytes ( e-g-
sodium chloride), stabilizers l e-g- 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
:
',
:. :
;Ds3a8N.Dcc

-~ 2~132~
47
according to the invention of the general formula (I), 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 (I) with a permetallate of a
radioactive metal ion, whereby a complex according to the
invention is formed containing the compound of general
formula (I) and the metal ion.
Furthermore, a cold kit is provided to prepare the
radiopharmaceuticals that contains endothelines,
endotheline derivatives, endotheline partial sequences,
endothe~ine analogues, or endotheline antagonists bonded
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 endotheline~, 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.
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, i
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.
~D933ilN~DOC
~': . t,. . ....

-- 21 ~ 32~ :
48
Another object of the invention is a method for imaging
pathological vascular changes characterized in that a
complex of a compound of general formula (I) 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 lodine
isotope. l3lI, l2sI, and l23I are particularly preferred.
These iodinated endothelines are prepared in a generally
known manner, preferably according to the chloramine-T
-~ocess, the lactoperoxidase proceSs the Bolton-Hunter
or the iodogen process
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.
In the accompanying drawings:
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 l23I 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 l23I endotheline 1.
~D9391iN.DOC

49 2~324~
The object of the invention is described in more detail
by the following examples.
Example 1
In-vivo and in-vitro accumulation of l23I endotheline 1
in WHHL rabbits.
Endotheline 1 was labelled with 123I using the
chloramine-T method (Krohn et al. 1977, BBA 490, 49). 20
~l endotheline 1 (2 ~g, equivalent to 0.8 nmol, in
phosphate buffer, 0.25 mol/l, pH 7.4; SIGMA Co.) are
added to 50 ~1 phosphate buffer (0.25 mol/l, pH 7.4), 100
~l of 123I (15 ~l 123I, Medgenix Co., 74 MBq, equivalent
to 2 mCi, brought to a total volume of 100 ~l by adding
0.01 mol/l of NaOH), and 100 ~l chloramine-T solution
(35.5 mmol/l phosphate buffer, 0.25 mol/l, pH 7.4) for 2
mins at room temperature. 1000 ~l sodium disulphite
~olution (Na2S2O5; 6.3 mmol/l, 0.25 mol/1, pH 7.4) are
added and briefly mixed to stop the reaction. 800 ~1
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 ~l). This reaction charge is gel filtrated
through Sephadex G-10 (mobile buffer: phosphate buffered
saline (7.4) 0.1 ~ BSA (w/v)). Labelling of endotheline
was successful at 39 ~.
The five fractions of l23I endotheline 1 with the highest
activity were 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
ID93 asN . DOC

21 ~ 32~
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 aftex
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 l23I (see Fig. 1). A
subsequent autoradiography revealed a complete labelling
of the aorta wall and the atherosclerotic plaques (see
Fig. 2). 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-asp-
ile-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 -15C and added to 29 mg
dicyclohexyl carbodiimide in absolute dimethyl formamide.
The reaction mixture is stirred for 2 hours at -5C, then
for 2 hours at room temperature, and then cooled to -
15C. N,N'-dicyclohexyl urea that settles down is
filtered off. The filtrate i9 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
ID933BU.DO~

~~~ 51 21~32~
organic solvent portion is blown off using N2, and the
residue is freeze-dried.
Molecular weight: calc. 1301.4
det. 1301 (FAB-MS)
b) 99mTc complex of S-benzoylthioacetyl-gly-gly-gly-
gly-asp-his-leu-asp-ile-ile-trp.
A solution of 0.5 mg of S-benzoylthioacetyl-gly-gly-gly-
gly-asp-his-leu-asp-ile-ile-trp in 300 ~1 phosphate
buffer (Na2HP04, 0.1 mol/l, pH 9.5) is added to 50 ~1 of
a 0.15 molar trisodium citrate dihydrate solution and 2.5
~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
for 15 min at room temperature and then filtrated (0.2
filter). Labelling is analysed using HPLC: MERCK
nucleosil-column, 125 x 4 mm, 5 ~m; gradient from 100 % A
to 100 ~ B within 7.5 min. EIuent A: phosphate buffer
tNa2HP04; 0.01 M; pH 2.0); eluent B:acetonitrile/
phosphate buffer (Na2HP04; 0.01 M; pH 2.0) 75:25 (V/V);
flow rate: 1.0 ml/min.
Example 3
a) N,N'-bis(S-benzoylthioacetyl)-3,4-diaminobutyryl-
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-
phe-cys-his-leu-asp-ile-ile-trp,
A solution of 50 mg N,N~-bis(S-benzoylthioacetyl)-3,4-
diaminobutyric acid and 15 mg N-hydroxysuccinimide in
absolute freshly distilled dimethyl formamide i5 cooled
to -15C and added to 28 mg dicyclohexyl carbodiimide in
absolute dimethyl formamide. The reaction mixture is
ID9331~1.DOC

` ` 2~32~
52
stirred for 2 hours at -5C, then for 2 hours at room
temperature, and then cooled to -15C. N,N'-dicyclohexyl
uxea 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-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 N2, and the residue is freeze-
dried.
Molecular weight: calc. 2948.5 ,;
det. 2949 (FAB-MS)
b) 99mTc complex of ~ ~`
N,N'-bis(S-benzoylthioacetyl)-3,4-diaminobutyryl-
.
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-
phe-cys-his-leu-asp-ile-ile-trp,
N,N'-bis~S-benzoylthioacetyl)-3-4-diaminobutyryl-cys-ser-
cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-cys-his-
leu-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 - I
cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-
I
ID93a8tl.WC
',','

2 ~ 3 3 2 -~ ~J
53
cys-his-leu-asp-ile-ile-trp.
. .
A solutio~n of 1 mg 4-carboxyethylphenylglyoxal-bis-(N-
methylthio semicarbazone)-N-hydroxy succinimide ester in
50 ~1 dioxane is added to a solution of 1 mg cys-ser-cys-
ser-ser-trp-leu-asp-lys-glu-cys-val-tyr-phe-cys-his-leu-
asp-ile-ile-trp (endotheline 2) in 100 ~1 phosphate
buffered saline, pH 8.4, in a nitrogen atmosphere, and
stirred for 15 hours at a temperature below 10C. 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 N2, and the residue is freeze-dried.
....
Molecular weight: calc. 2895.4
det. 2894 (FAB-MS)
b) 99mTc complex of
3-[p-phenylglyoxal-di(N-methylthio semicarbazone)]-
propionyl-cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-
I~ ,
val-tyr-phe-cys-his-leu-asp-ile-ile-trp.
3-[p-phenylglyoxal-di-(N-methylthio semicarbazone)]-
propionyl-cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys- ~'
val-tyr-phe-cys-his-leu-asp-ile-ile-trp is labelled
according to example 2b.
Example 5
a) 3,6-Diaza-1,8-dimercapto-2,7-bis-(carbonyl-gly-his-
leu-asp-ile-ile-trp) octane.
ID9~8N . I~OC

` 54 21i324~
256 mg (0.5 mmol) N,N~-1,2-ethylene diyl-bis-L-cysteine-
di-(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) NH2-gly-his-leu-asp-ile-ile-
trp (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., Roc~ford, 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
residue is mixed up with 200 ml absolute diethyl ether.
The white solid that settles down is filtered off. It is ~;
recrystalliæed from dimethyl formamide/diethyl ether
mixtures.
.
Yield: 278 mg (28.7 %), white powder.
Analysis: calculated on the anhydrous substance
Calc.: C 55.77 H 6.86 N 15.90 O 18.16 S 3.31
Found: C 55.49 H 7.03 N 15.81 S 3.07
b) 99mTc 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-1,8-
dimercapto-2,7-bis-(carbonyl-gly-his-leu-asp-ile-ile-trp)
octane in 300 ~1 phosphate buffer (Na2HPO4, 0.5 mol/l, pH
7.0) is added to 50 ~l of a 0.15 molar trisodium citrate
dihydrate solution and 2.5 ~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 for 10 min at room
temperature and then filtrated (0.2 ~ filter). Labelling
~D9331~N.NC

55 2 1 1 3 2 4 ~
is analysed using HPLC: MERCK nucleosil-column, 125 x 4
mm, 5 ~m; gradient from loO ~ A to 100 ~ B within 7.5 -
min. Eluent A: phosphate buffer (Na2HP04); 0.01 M; pH
2.0) 50 : 50 (V/V); flow rate: 1.0 ml/min.
Example 6
a) {[(3,3,9,9-tetramethyl-4,8-diaza-undecyl-2,10-dion-
dioxime?-6-yl]methylphen-4~-yl} aminothiocarbonyl-cys-
,~,
~er-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-
his-leu-asp-ile-ile-trp.
A solution of 1 mg 6-(4'-isothiocyanatobenzyl)-3,3,9,9-
tetramethyl-4,8-diazaundecane-2,10-dion-dioxime in 50 ~1
tetrahydrofurane is mixed in a nitrogen atmosphere with a
solution of 1 mg cys-ser-ala-ser-ser-leu-met-asp-lys-glu-
ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp (ala-
endotheline) in 100 ~1 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 N2, and the residue is freeze-dried.
Molecular weight: calc. 2833.3
det. 2833 (FAB-MS)
b) 99mTc complex of {[(3,3,9,9-tetramethyl-4,8-diaza-
undecyl-2,10-dion-dioxime)-6-yl]methylphen-4'-yl}
aminothiocarbonyl-cys-ser-ala-ser-ser-leu-met-asp-lys-
glu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp.
{[(3,3,9,9-tetramethyl-4,8-diaza-undecyl-2,10-dion-
dioxime)-6-yl]methylphen-4'-yl} aminothiocarbonyl-cys-
ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-
ID93 311N . DOI:

` 56 2~ 132~
his-leu-asp-ile-ile-trp is labelled according to example
2b.
Example 7 ;~
a) N,N'-bis(S-benzoylthioacetyl)-4,5-diamino-1-oxo-
..
pentyl-cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-
tyr-phe-cys-his-leu-asp-ile-ile-trp. ;~
A solution of 50 mg N,N'-bis(S-benzoylthioacetyl)-4,5-
diaminopentanoic acid and 15 mg N-hydroxy succinimide in
absolute freshly distilled dimethyl formamide is cooled
to -15C and added to 28 mg dicyclohexyl carbodiimide in ~ ;~
absolute dimethyl formamide. The reaction mixture is
stirred for 2 hours at -5C, then for 2 hours at room ~`
temperature, and then cooled to -15C. 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-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 re ults in a flocculent
precipitate which i9 isolated and then purified using
preparative HPLC (gradient: acetonitrile/phosphate
buffer). After neutralizing the buffered solution, the
organic solvent portion is blown off using N2, and the
residue is freeze-dried.
Molecular weight: calc. 2962.5
det. 2963 (FAB-MS)
~D911RN.DOC

- ` 2~ ~32~
- 57
b) 99mTc complex of N,N~-bis(S-benzoylthioacetyl)-4,5-
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.
N,N'-bis(S-benzoylthioacetyl)-4,5-diamino-1-oxo-pentyl-
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-
cys-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} aminothiocarbonyl-
r
cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr-
tyr-cys-his-leu-asp-ile-ile-trp. `~
A solution of 1 mg 2-methyl-2-(4-isothiocyanatobenzyl)-
N,N'propylene-bis-salicylidenamine in 50 ~1
tetrahyd~rofurane is mixed in a nitrogen atmosphere with a
solution of 1 mg cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-
cys-val-tyr-tyr-cys-his-leu-asp-ile-ile-trp (endotheline
3) in 100 ~l water and stirred overnight. The conjugate
is purified using a preparative HPLC (gradient:
acetonitrile/phosphate buffer). After neutralizing the
buffered solution, the organic sol~ent portion is blown
off using N2, and the residue is freeze-dried.
Molecular weight: calc. 3090.7
det. 3091 ~F~3-MS)
b) 99mTc complex of {[2,6-diaza-1,7-di(2-hydroxyphenyl)-
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-trp.
. .
ID93 38N . DOC
~,. .

- 21~2~ ~
58
{[2,6-diaza-1,7-di(2-hydroxyphenyl)-4-methyl-hept-4-yl]-
methyl-phen-4'-yl~ aminothio-carbonyl-cys-thr-cys-phe-
thr-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)-4-
methyl-hept-4-yl]-methylphen-4'-yl} aminothiocarbonyl-
ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe- ;
ala-his-leu-asp-ile-ile-trp. ~-
A fresh solution of 1 mg 2-methyl-2-(4-
isothiocyanatobenzyl)-N,N'-propylene-bis-[5-
(sulpho)salicylidenamine3 in 50 ~1 water is mixed in a
nitrogen atmosphere with a solution of 1 mg ala-ser-ala-
ser-ser-ieu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-
asp-ile-ile-trp in 100 ~l 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 N2, and the residue is freeze-
dried.
Molecular weight: calc. 2959.4
det. 2959 (FAB-MS)
b) 99mTc complex of ~[2,6-diaza-1,7-di(2-hydroxy-5-
sulphophenyl)-4-methyl-hept-4-
yl]-methylphen-4'-yl} aminothiocarbonyl-ala-ser-ala-ser-
ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-
ile-ile-trp.
{[2,6-diaza-1,7-di(2-hydroxy-5-sulphophenyl)-4-methyl-
hept-4-yl]-methylphen-4~-yl} aminothiocarbonyl-ala-ser-
: :
ID9338N~WC
. - ~ . - .. ; . - ~ ~ . ~. ~ . . . ,:: . :

59 2~32~
ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-
leu-asp-ile-ile-trp is labelled according to example 2b.
Example 10
a) N-[3,6,9-triaza-1-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-(2,6-
dioxomorpholino)-ethyl]-3,6-diaza-octanedioic acid (DTPA-
monoethyl ester monoanhydride) is added in portions to a
solution of 853 mg (1 mmol) NH2-gly-his-leu-asp-ile-ile-
trp (prepared in analogy to Barany and Marrifield, The
Peptides: Analysis, Biology, Academic Preqs, 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
temperature. When the reaction is finished, the mixture
i8 filtrated, and the solvent is drawn off under vacuum.
To remaining oil 50 ml diethylformamide 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 dimethyl
formamide/diethyl ether mixtures.
Yield: 634 mg (50.4 %), white powder.
Analysis: calculated on the anhydrous substance
Calc.: C 54.49 H 6.82 N 14,49 O 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-ile-
trp. -
.~.,
~D9311EN.DOC

2 ~ .5
1.26 mg (1 mmol) of the prepared N-[3,6,9-triaza-1-oxo-
3,6,9-tris-(hydroxycarbonyl-methyl)-9-
ethoxycarbonylmethyl)-nonyl]-gly-his-leu-asp-ile-ile-trp
(example lOa) 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
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.
Yield: 213 mg (17.3 %), white powder.
Analy~is: 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-ile-
trp (example lOb) can also be prepared by reacting 853 mg
(1 mmol) NH2-gly-his-leu-asp-ile-ile-trp (example lOa),
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 lN
hydrous sodium hydroxide solution. Then, a pH value of 2
is set by adding concentrated hydrochloric acid. The
crude con~ugate is filtered off and rewashed using a
~D93311N.DOC

61 21 l32~
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 4.5.
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 ) 99mTc complex of N-[3,6,9-triaza-1-oxo-3,6,9,9-tetra-
(hydroxycarbonylmethyl)-nonyl]-gly-his-leu-asp-ile-ile-
trp
A solution of 0.5 mg of the prepared N-~3,6,9-triaza-1-
oxo-3,6,9,9-tetra-(hydroxy-carbonylmethyl)-nonyl]-gly-
his-leu-asp-ile-ile-trp (example lOb) in 300 ~l phosphate
buffer (Na2HP04, 0.5 mol/l, pH 7.0) is added to 50 ~1 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
filter). Labelling is analysed using HPLC: MERCK
nucleosil-coulmn 125 x 4 mm, 5 ~m; gradient from 100 ~ A
to 100 ~ B within 7.5 min. Eluent A: phosphate buffer -
(Na2HP04); 0.01 M; pH 2.0) 50 : 50 (V/V); flow rate: 1.0
ml/min.
d) lllIn complex of N-[3,6,9-triaza-1-oxo-3,6,9,9-tetra-
(hydroxycarbonylmethyl)-nonyl]-gly-his-leu-asp-ile-ile-
trp
ID9391~N.DOC

62 2~ ~3 2
A solution of 0.5 mg of the prepared N-[3,6,9-triaza-1-
oxo-3,6,9,9-tetra-(hydroxy-carbonylmethyl)-nonyl]-gly-
his-leu-asp-ile-ile-trp (example lOb) in 300 ~l phosphate
buffer (Na2HPO4, 0.5 mol/l, pH 6.5) is incubated for 10
min at room temperature using 20 ~ 1InCl3 (3 MBq, NEN,
Du Pont) and then filtrated (0.2 ~ filter). Labelling is
analysed using HPLC: MERCK nucleosil-column 125 x 4 mm, 5
~m; gradient from 100 % A to 100 ~ B within 7.5 min.
Eluent A: phosphate buffer (Na2HPO4); 0.01 M; pH 2.0) 50
: 50 (V/V); flow Fate: 1.0 ml/min.
e) Gd(III) complex of N-[3,6,9-triaza-1-oxo-3,6,9,9-
tetra-(hydroxycarbonylmethyl)-nonyl]-gly-his-leu-asp-ile-
ile-trp, sodium salt.
669 mg (2 mmol) gadolinium(III) acetate is added in
portion at a pH of 6.5 to a solution of 2.46 g (2 mmol)
of the prepared N-[3,6,9-triaza-1-oxo-3,6,9,9-tetra-
(hydroxy-carbonylmethyl)-nonyl]-gly-his-leu-asp-ile-ile-
trp (example lOb) in 100 ml water while keeping the pH ofthe 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
ID9391~N.DOC

` ` 63 2~~~2~
Calc.: C 46.31 H 5.37 N 12.76 O 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-asp-
ile-ile-trp-thiouridyl)phenoxy] propyl} 1,4,7,10-
tetraaza-4,7,10-tris-(carboxylatomethyl)-cyclododecane
708 mg (1 mmol) 1-[2-hydroxy-3-(4-isothiocyanato-
phenoxy)-propyl]-1,4,7,10-tetraaza-4,7,10-tris-
(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) NH2-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
reaction is finished, the mixture is filtrated, and the ~ ;
solvent is drawn off under vacuum. To the remaining oil
5Q 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 i9 recrystallized from
dimethylformamide/d;.ethyl 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
ID9331~N.DOC
. ' '
',',~'~:! ` :, ' , :, '- :. :`,:: . .~ . . ` , ' ' ' .'.:, . . :

64 2t ~324~
Found: C 49.78 H 6.23 N 13.17 S 1.89 Gd 9.83
Example 12
a) cys(acm)-gly-cys(acm)-
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr- ~-
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 -15C and added to 28 mg ~;
dicyclohexyl carbodiimide in absolute dimethylformamide.
The reaction mixture is stirred for 2 hours at -5C, then
for 2 hours at room temperature, and then cooled to -
15C. 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-
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
30 min, and subsequently concentrated to a minimum under
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 N2, and the residue is freeze-dried.
Molecular weight: calc. 2897.4
¦ det. 2898 (FAB-MS)
ID93 911N . DOC
.

~ ~3~ ~
b) 99mTc complex of cys(acm)-gly-cys(acm)-
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-
phe-cys-his-leu-asp-ile-ile-trp.
Cys(acm)-gly-cys(acm)-cys-ser-cys-ser-ser-leu-met-asp-
lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp is
labelled according to example 2b.
1 0
Example 13
a) Cyclo(trp-leu-val-pro-asp)-cys(acm)-gly-cys(acm). -
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 -15C and added to a
solution of 51.7 mg 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride in 8 ml absolute dimethyl ~-
formamide. The reaction mixture is stirred for 2 hours at ~-
-5C, 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 N2, and the residue is freeze-
dried.
Molecular weight: calc. 1016.2
det. 1016 (FAB-MS)
: .
~, ' ' .
. -,.:
ID9331~N.DOC

66 21 13~ ~ ~
b) 99mTc 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-met-
asp-lys-glu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp
A solution of 500 ~g cys-ser-ala-ser-ser-leu-met-asp-lys-
glu-ala-val-tyr-phe-cys-his-leu-asp-ile-ile-trp (ala-
endotheline) in 400 ~l 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 ~l triethanolamine hydrochloride buffer
pH 8.0). Then the reaction is finished by separating
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)
b) 99mTc 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
A solution of 0.5 mg of 4-mercaptobutyrimidyl-cys-ser-
ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-cys-his-
leu-asp-ile-ile-trp in 300 ~1 phosphate buffer (Na2HPO4,
0.1 mol/l, pH 9.5) is added to 50 ~l of a 0.15 molar
trisodium citrate dihydrate solution and 2.5 ~1 of a 0.2
molar tin(II) chloride dihydrate solution. The reaction
~D933RN.DOC
!::

67 2~ 132~
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 ~ filter).
Labelling is analysed using HPLC: MERCK nucleosil-column,
125 x 4 mm, 5 ~m; gradient from 100 % A to 100 ~ B within
7.5 min. Eluent A: phosphate buffer (Na2HPO4; 0.01 M; pH
2.0); eluent B: acetonitrile/ phosphate buffer (Na2HPO4; -
0.01 M; pH 2.0) 75:25 (V/V); flow rate: 1.0 ml/min.
Example 15
a) 3-Thiopropionyl-gly-asp-his-leu-asp-ile-ile-trp. ~;
A ~olution of 500 ~g gly-asp-his-leu-asp-ile-ile-trp in
400 ~1 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
i~ then reduced using 25 mM dithiothreitol, pH 4.5. The
reaction solution is concentrated under vacuum to a
minimum. Then it is purified using preparative HPLC
~gradient: acetonitrile/phosphate buffer). After
neutralizing the buffered solutior., the organic solvent
portion i5 blown off using N2, and the residue is freeze-
dried.
Molecular weight: calc. 1040.2
det. 1040 (FAB-MS)
b) 99mTc complex of 3-Thiopropionyl-gly-asp-his-leu-
asp-ile-ile-trp.
3-Thiopropionyl-gly-asp-his-leu-asp-ile-ile-trp is
labelled according to example 2b.
109~-D.I:C

2~132~
68
Example 16
a) 2-(Acetylthio)succinyl-gly-asp-his-leu-asp-ile-ile-
trp.
A solution of 500 ~g gly-asp-his-leu-asp-ile-ile-trp in
50 ~l dimethyl formamide is added to a 10 times molar
excess of 2-mercaptoacetyl succinic acid anhydride in
5 ~l dimethylformamide and incubated for 2 hours at room
temperature. Then it is purified using preparative HPLC
(gradient: acetonitrile/phosphate buffer). The organic
solvent portion is blown off using N2, and the residue is
freeze-dried.
Molecular weight: calc. 1126.2
det. 1126 (FAB-MS)
b) 99mTc complex of 2-(Acetylthio)succinyl-gly-asp-his-
leu-asp-iile-ile-trp.
2-(Acetylthio)~uccinyl-gly-asp-his-leu-asp-ile-ile-trp iB
labelled according to example 2b.
Example 17
a) Reduction of
cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr-phe-
L~
cys-his-leu-asp-ile-ile-trp (endotheline 1)
A solution of 0.5 mg cys-ser-cys-ser-ser-leu-met-asp-lys-
glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp in 500 ~l
phosphate buffered saline (pH 7.4) is mixed with 18 ~l
mercapto ethanol and incubated for 30 min at room
temperature; the excessive reductant is separated.
~D93UN . DOC

69 2 1 l 3 2 ~
b) 99mTc complex of cys-ser-cys-ser-ser-leu-met-asp~
lys-glu-cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp
A solution of 0.5 mg of the reduced endotheline 1 in 300
~l phosphate buffer (Na2HPO4, 0.5 mol/l, pH 9.5) i9 added ~ -~
to 50 ~l 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 ~ filter). Labelling is analy~ed
using HPLC: MERCK nucleosil-column, 125 x 4 mm, 5 ~m;
gradient from 100 ~ A to 100 ~ B within 7.5 min. Eluent
A: phosphate buffer (Na2HPO4; 0.01 M; pH 2.0); eluent B:
acetonitrile/phosphate buffer (Na2HPO4; 0.01 M; pH 2.0)
50 : 50 (V/V); flow rate: 1.0 ml/min.
IW33BN.DOC