U.S. patent application number 11/390472 was filed with the patent office on 2007-01-11 for macrocyclic metal complexes and their use for the production of conjugates with biomolecules.
Invention is credited to Hans Bauer, Thomas Frenzel, Gunther Michl, Johannes Platzek, Bernd Raduchel, Heiko Schirmer, Heribert Schmitt-Willich, Detlev Sulzle, Hanns-Joachim Weinmann.
Application Number | 20070009442 11/390472 |
Document ID | / |
Family ID | 7692471 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070009442 |
Kind Code |
A1 |
Platzek; Johannes ; et
al. |
January 11, 2007 |
Macrocyclic metal complexes and their use for the production of
conjugates with biomolecules
Abstract
The invention relates to macrocyclic metal complexes and their
production and use for the production of conjugates with
biomolecules. The conjugates are suitable as contrast media in NMR
diagnosis and radiodiagnosis as well as for radiotherapy. High
relaxivity is achieved by a special liganding of macrocyclic
compounds, and a fine-tuning of the relaxivity is made
possible.
Inventors: |
Platzek; Johannes; (Berlin,
DE) ; Schmitt-Willich; Heribert; (Berlin, DE)
; Michl; Gunther; (Rudersdorf, DE) ; Frenzel;
Thomas; (Berlin, DE) ; Sulzle; Detlev;
(Berlin, DE) ; Bauer; Hans; (Berlin, DE) ;
Raduchel; Bernd; (Berlin, DE) ; Weinmann;
Hanns-Joachim; (Berlin, DE) ; Schirmer; Heiko;
(Berlin, DE) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD.
SUITE 1400
ARLINGTON
VA
22201
US
|
Family ID: |
7692471 |
Appl. No.: |
11/390472 |
Filed: |
March 28, 2006 |
Current U.S.
Class: |
424/9.34 ;
530/409; 536/25.32; 536/53; 540/145; 540/474 |
Current CPC
Class: |
A61K 49/10 20130101;
A61K 49/0002 20130101; A61K 49/085 20130101; A61K 47/547 20170801;
A61K 49/14 20130101; A61K 49/143 20130101; A61K 47/54 20170801;
C07D 257/02 20130101 |
Class at
Publication: |
424/009.34 ;
540/474; 530/409; 536/025.32; 536/053; 540/145 |
International
Class: |
A61K 49/10 20060101
A61K049/10; C07K 14/47 20060101 C07K014/47; C07H 21/04 20060101
C07H021/04; C07D 257/02 20060101 C07D257/02; C07D 487/22 20060101
C07D487/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 20, 2001 |
DE |
10135356.1 |
Claims
1. Compounds of formula I ##STR18## in which Z represents a
hydrogen atom or at least two Z's represent a metal ion equivalent,
B.sup.1, B.sup.2, B.sup.3, B.sup.4 are independently selected from
the group consisting of hydrogen atoms and C.sub.1-4-alkyl
radicals, R.sup.1, R.sup.2, R.sup.3 are independently selected from
the group consisting of hydrogen atoms and straight, branched or
cyclic, saturated or unsaturated C.sub.1-10-alkyl or aryl radicals,
which optionally are substituted with a carboxyl group --SO.sub.3H
or --PO.sub.3H.sub.2, and whereby the alkyl chains of the
C.sub.1-10-alkyl radicals optionally contain an aryl group and/or
1-2 oxygen atoms, provided that at least one of the radicals
B.sup.1, B.sup.2, B.sup.3, B.sup.4, R.sup.1, R.sup.2 and R.sup.3
does not represent a hydrogen atom, A represents a straight or
branched, saturated or unsaturated C.sub.1-30-hydrocarbon chain
that optionally contains 1-5 oxygen atoms, 1-5 nitrogen atoms
and/or 1-5 --NR' radicals, in which R' is defined as R.sup.1,
R.sup.2 and R.sup.3 but can be selected independently, which
optionally is substituted with 1-3 carboxyl groups, 1-3
--SO.sub.3H, 1-3 --PO.sub.3H.sub.2 and/or 1-3 halogen atoms, in
which optionally 1-3 carbon atoms are present as carbonyl groups,
whereby the chain or a portion of the chain can be arranged
concentrically, and which is configured in such a way that X is
connected via at least 3 atoms to the nitrogen to which A is
bonded, and X represents a group that can participate in a reaction
with a biomolecule, as well as their salts, provided that a) If
B.sup.1, B.sup.2, B.sup.3 and B.sup.4 are hydrogen atoms and
R.sup.1, R.sup.2 and R.sup.3 are --CH.sub.2CH.sub.2CO.sub.2H, A-X
together are not --CH(CO.sub.2H)CH.sub.2CH.sub.2CO.sub.2H, b) If
B.sup.1, B.sup.2, B.sup.3 and B.sup.4 are hydrogen atoms and
R.sup.1, R.sup.2 and R.sup.3 are methyl or R.sup.1, R.sup.2 and
R.sup.3 are ethyl radicals, which optionally are substituted with a
carboxy group, A does not represent the radical
--CH(R.sup.7)--CO--NR.sup.5U.sup.6--, in which R.sup.5 stands for a
hydrogen atom, a methyl or an ethyl radical, which optionally is
substituted with 1 carboxy group, R.sup.7 stands for a
straight-chain, branched, saturated or unsaturated
C.sub.1-C.sub.30-alkyl chain, which optionally is interrupted by
1-10 oxygen atoms, I phenylene group, or 1-phenylenoxy group,
and/or optionally is substituted by 1-5 hydroxy groups, 1-3 carboxy
groups or 1 phenyl group, and U.sup.6 stands for a straight-chain,
branched, saturated or unsaturated C.sub.1-C.sub.20-alkylene group
that contains 1-5 imino groups, 1-3 phenylene groups, 1-3
phenylenoxy groups, 1-3 phenylenimino groups, 1-5 amide groups, 1-2
hydrazide groups, 1-5 carbonyl groups, 1-5 ethylenoxy groups, 1
urea group, 1 thiourea group, 1-2 carboxyalkylimino groups, 1-2
ester groups, 1-10 oxygen atoms, 1-5 sulfur atoms and/or 1-5
nitrogen atoms and/or is optionally substituted by 1-5 hydroxy
groups, 1-2 mercapto groups, 1-5 oxo groups, 1-5 thioxo groups, 1-3
carboxy groups, 1-5 carboxyalkyl groups, 1-5 ester groups and/or
1-3 amino groups, whereby the optionally contained phenylene groups
can be substituted by 1-2 carboxy groups, 1-2 sulfone groups or 1-2
hydroxy groups, and c) If B.sup.1, B.sup.2, B.sup.3 and B.sup.4 are
hydrogen atoms and R.sup.1, R.sup.2 and R.sup.3 represent the same
C.sub.1-4-alkyl radical, A does not represent the radical ##STR19##
in which R.sup.6 is a hydrogen atom or a C.sub.1-4-alkyl radical, D
is a saturated or unsaturated, straight-chain or branched
C.sub.1-4-alkylene group, which optionally can be interrupted or
substituted with a carbonyl group, and D is bonded to X.
2. Compounds according to claim 1, in which R.sup.1, R.sup.2 and
R.sup.3 are independently selected from the group consisting of
hydrogen atoms, straight-chain or branched C.sub.1-10-alkyl
radicals, cyclohexyl radicals, --CH.sub.2--COOH,
--C(CH.sub.3).sub.2--COOH, phenyl radicals or radicals of formula
--(CH.sub.2).sub.m--(O).sub.n-(phenylene).sub.p-Y, in which m is an
integer from 1 to 5, n is 0 or 1, p is 0 or 1, and y represents a
hydrogen atom, a methoxy radical, a carboxyl group, --SO.sub.3H or
--PO.sub.3H.sub.2.
3. Compounds according to claim 2, in which if B.sup.1, B.sup.2,
B.sup.3 and B.sup.4 are hydrogen atoms, R.sup.1, R.sup.2 and
R.sup.3 are independently selected from the group consisting of
hydrogen atoms, isopropyl radicals, isobutyl radicals, tert-butyl
radicals, straight-chain or branched C.sub.5-10-alkyl radicals,
cyclohexyl radicals, --CH.sub.2--COOH, --C(CH.sub.3).sub.2--COOH,
phenyl radicals or radicals of formula
--(CH.sub.2).sub.m--(O).sub.n-(phenylene).sub.p-Y, in which m is an
integer from 1 to 5, n is 0 or 1, p is 0 or 1, and Y represents a
hydrogen atom, a methoxy radical, a carboxyl group, --SO.sub.3H or
--PO.sub.3H.sub.2, provided that at least one of the radicals
R.sup.1, R.sup.2 and R.sup.3 does not represent a hydrogen
atom.
4. Compounds according to claim 3, in which if B.sup.1, B.sup.2,
B.sup.3 and B.sup.4 are hydrogen atoms, R.sup.1, R.sup.2 and
R.sup.3 are independently selected from the group consisting of
hydrogen atoms, isopropyl, cyclohexyl or phenyl radicals, provided
that at least one of the radicals R.sup.1, R.sup.2 and R.sup.3 does
not represent a hydrogen atom.
5. Compounds according to claim 1, in which A represents a radical
A'-U, in which A' is bonded to the nitrogen atom of the macrocyclic
ring and U is bonded to X, and whereby A' represents a) A bond, b)
--CH(CO.sub.2H)--, c) a group of formula ##STR20## in which Q
represents a hydrogen atom, a C.sub.1-10-alkyl radical, which
optionally is substituted with a carboxyl group, or an aryl
radical, which optionally is substituted with a carboxyl group, a
C.sub.1-15-alkoxy group, an aryloxy group or a halogen atom, and R'
is defined as R.sup.1, R.sup.2 and R.sup.3 in claim 1, but can be
selected independently, or d) a group of formula ##STR21## in which
o is 0 or 1, and the ring optionally is annellated with a benzene
ring, whereby this benzene ring, if present, can be substituted
with a methoxy or carboxyl group, --SO.sub.3H or --PO.sub.3H.sub.2,
whereby in the groups under c) and d), the positions that are
marked ##STR22## are bonded to the adjacent groups, and in which
position .alpha. is bonded to a nitrogen atom of the macrocyclic
ring and position .beta. is bonded to U, and U represents a
straight or branched, saturated or unsaturated
C.sub.1-30-hydrocarbon chain that optionally contains 1-3 oxygen
atoms, 1-3 nitrogen atoms and/or 1-3 --NR'' radicals, in which R''
is defined as R.sup.1, R.sup.2 and R.sup.3 in claim 1 but can be
selected independently, and in which optionally 1-3 carbon atoms
are present as carbonyl groups, whereby the chain or a portion of
the chain can be arranged concentrically, provided that A' and U
together are configured in such a way that X is bonded via at least
3 atoms with the nitrogen atom to which A' is bonded.
6. Compounds according to claim 5, in which for A', the group of
formula ##STR23## is selected from --C(CH.sub.3)H--CO--NH--,
--C(phenyl)H--CO--NH-- and --C(p-dodecanoxyphenyl)H--CO--NH--.
7. Compounds according to claim 5, in which for A', the group of
formula ##STR24## is selected from: ##STR25## whereby R.sup.4 is
--OCH.sub.3, --CO.sub.2--H, --SO.sub.3H or --PO.sub.3H.sub.2.
8. Compounds according to claim 5, in which U is selected from
--CH.sub.2--, --(CH.sub.2).sub.5--, --(CH.sub.2).sub.10--,
-phenylene-O--CH.sub.2--, -phenylene-O--(CH.sub.2).sub.3--,
-phenylene-O--(CH.sub.2).sub.10--, --CH.sub.2-phenylene-,
-cyclohexylene-O--CH.sub.2--, -phenylene-, --C(phenyl)H--,
--CH.sub.2-pyridylene-O--CH.sub.2--, --CH.sub.2-pyridylene- and
--CH.sub.2--CO--NH--CH.sub.2--CH.sub.2--
9. Compounds according to claim 1, in which X is selected from the
group that consists of carboxyl, activated carboxyl, amino,
isocyanate, isothiocyanate, hydrazine, semicarbazide,
thiosemicarbazide, chloroacetamide, bromoacetamide, iodoacetamide,
acylamino, mixed anhydrides, azide, hydroxide, sulfonyl chloride,
carbodiimide and radicals of formulas ##STR26## in which Hal is a
halogen atom.
10. Compounds according to claim 9, in which the activated carboxyl
group is selected from ##STR27##
11.
10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alph-
a.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)
1,4,7-.alpha.,.alpha.',.alpha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl-
)-1,4,7,10-tetraazacyclododecane,
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-
-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-
,
10-(4-(t-butoxycarbonyl-1-phenyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.-
',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane,
10-[.alpha.-(4-(ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-
-1,4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,-
7,10-tetraazacyclododecane,
10-[.alpha.-(4-(ethoxycarbonylpropoxy)phenyl)-methoxycarbonylmethyl]-1,4,-
7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10--
tetraazacyclododecane,
10-[.alpha.-(4-ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1,4,-
7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10--
tetraazacyclododecane,
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tri-
s(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(isopropyl)-1,4-
,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(cyclohexyl)-1,-
4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-triphenyl-1,4,7-tri-
s(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-(4-(t-butoxycarbony-1-phenyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',-
.alpha.''-triphenyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododeca-
ne,
10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(-
isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(cyc-
lohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-2,5,8,11-tetramethyl-1,4,7,10-te-
traazacyclododecane-1,4,7-triacetic acid-tri-tert-butyl ester,
10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-.alpha.,.alp-
ha.',.alpha.''-tris-(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraaz-
acyclododecane and
10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-.alpha.,.alp-
ha.',.alpha.''-tris(cyclohexyl)
1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.alph-
a.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.alph-
a.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
ane,
10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.-
alpha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-
dodecane,
10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.al-
pha.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-
dodecane,
10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.al-
pha.',.alpha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraaz-
acyclododecane,
10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodo-
decane,
10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.'-
,.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
ane,
10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.a-
lpha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodo-
decane,
10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.'-
,.alpha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyc-
lododecane,
10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,
10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane,
10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.'-
,.alpha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyc-
lododecane,
10-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-.alpha.,.alpha.'-
-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-isopropyl-4-benzyloxyc-
arbonylmethyl-1,4,7,10-tetraazacyclododecane,
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-.alpha.,.alpha.'-
-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-methyl-4-benzyloxyc-
arbonylmethyl-1,4,7,10-tetraazacyclododecane,
10-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-.alpha.,.alpha.'-
-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-cyclohexyl-4-benzyloxy-
carbonylmethyl-1,4,7,10-tetraazacyclododecane,
10-[.alpha.-(4-(ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,7--
.alpha.,.alpha.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-isopro-
pyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,
10-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-.alpha.-.alpha.'-diisopro-
pyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-methyl-4-benzyloxycarbonylme-
thyl-1,4,7,10-tetraazacyclododecane,
10-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-.alpha.-.alpha.'-dimethyl-
-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-cyclohexyl-4-benzyloxycarbonylm-
ethyl-1,4,7,10-tetraazacyclododecane.
12. Compounds according to claim 1, in which at least two of
radicals Z stand for a metal ion equivalent of a radioactive or
paramagnetic element of atomic numbers 21-29, 31, 32, 37-39, 42-44,
46, 47, 49, 58-71, 75, 77, 82 or 83.
13. Use of the compounds of formula I ##STR28## in which Z,
B.sup.1, B.sup.2, B.sup.3, B.sup.4, R.sup.1, R.sup.2, R.sup.3, A
and X are defined as in claim 1, provided that at least one of the
B.sup.1, B.sup.2, B.sup.3, B.sup.4, R.sup.1, R.sup.2 and R.sup.3
does not represent a hydrogen atom and if B.sup.1, B.sup.2,
B.sup.3, B.sup.4 are hydrogen atoms and R.sup.1, R.sup.2, R.sup.3
represent the same C.sub.1-4-alkyl radicals, A does not represent
the radical ##STR29## in which R.sup.6 is a hydrogen atom or a
C.sub.1-4-alkyl radical, D is a saturated or unsaturated,
straight-chain or branched C.sub.1-4-alkylene group, which
optionally is interrupted or substituted with a carbonyl group, and
D is bonded to X, for the production of a conjugate with a
biomolecule.
14. Use according to claim 13, in which the biomolecule is selected
from the group that consists of biopolymers, proteins,
synthetically modified biopolymers, carbohydrates, antibodies, DNA
and RNA fragments, .beta.-amino acids, vector amines for transfer
into the cell, biogenic amines, pharmaceutical agents, oncological
preparations, synthetic polymers, which are directed to a
biological target, steroids, prostaglandins, taxol and derivatives
thereof, endothelins, alkaloids, folic acid and derivatives
thereof, bioactive lipids, fats, fatty acid esters, synthetically
modified mono-, di- and triglycerides, liposomes, which are
derivatized on the surface, micelles that consist of natural fatty
acids or perfluoroalkyl compounds, porphyrins, texaphrines,
expanded porphyrins, cytochromes, inhibitors, neuramidases,
neuropeptides, immunomodulators, endoglycosidases, substrates that
are attacked by the enzymes calmodulin kinase, casein-kinase II,
glutathione-S-transferase, heparinase, matrix-metalloproteases,
.beta.-insulin-receptor-kinase, UDP-galactose 4-epimerase,
fucosidases, G-proteins, galactosidases, glycosidases,
glycosyltransferases and xylosidase, antibiotics, vitamins and
vitamin analogs, hormones, DNA intercalators, nucleosides,
nucleotides, lectins, vitamin B12, Lewis-X and related substances,
psoralens, dienetriene antibiotics, carbacyclins, VEGF,
somatostatin and derivatives thereof, biotin derivatives,
antihormones, tumor-specific proteins and synthetic agents,
polymers that accumulate in acidic or basic areas of the body,
myoglobins, apomyoglobins, neurotransmitter peptides, tumor
necrosis factors, peptides that accumulate in inflamed tissues,
blood-pool reagents, anion and cation-transporter proteins,
polyesters, polyamides and polyphosphates.
15. Process for the production of compounds of formula I ##STR30##
in which Z, B.sup.1, B.sup.2, B.sup.3, B.sup.4, R.sup.1, R.sup.2,
R.sup.3, A and X are defined as in claim 1, provided that at least
one of the B.sup.1, B.sup.2, B.sup.3, B.sup.4, R.sup.1, R.sup.2 and
R.sup.3 does not represent a hydrogen atom and if B.sup.1, B.sup.2,
B.sup.3, B.sup.4 are hydrogen atoms and R.sup.1, R.sup.2, R.sup.3
represent the same C.sub.1-4-alkyl radicals, A does not represent
the radical ##STR31## in which R.sup.6 is a hydrogen atom or a
C.sub.1-4-alkyl radical, D is a saturated or unsaturated,
straight-chain or branched C.sub.1-4-alkylene group, which
optionally is interrupted or substituted with a carbonyl group, and
D is bonded to X, in which a compound of formula II ##STR32## in
which B.sup.1, B.sup.2, B.sup.3 and B.sup.4 are defined as in claim
1, is optionally reacted with Nu-A-X', Nu-CH(R.sup.1)--CO.sub.2-Z',
Nu-CH(R.sup.2)--CO.sub.2-Z' and Nu-CH(R.sup.3)--CO.sub.2-Z'
optionally after protective groups for the nitrogen atoms are
introduced, whereby A and R.sup.1, R.sup.2 and R.sup.3 are defined
as in claim 1, and Nu is a nucleofuge, X' stands for X or a
protected form of X, and X is defined as in claim 1, and Z' stands
for a hydrogen atom, a metal ion equivalent or a protective group
for carboxyl, then the optionally present protective groups are
removed, and, if desired, reacted in a way that is known in the art
with at least one metal oxide or metal salt of a desired element
and optionally then still present acid hydrogen atoms are
completely or partially substituted in the thus obtained complexes
by cations of inorgnaic and/or organic bases, amino acids or amino
acid amides.
Description
[0001] The invention relates to the subjects that are characterized
in the claims, i.e., macrocyclic metal complexes as well as their
use for the production of conjugates with biomolecules. The
conjugates are suitable for the production of contrast media for
NMR diagnosis and radiodiagnosis as well as agents for
radiotherapy.
[0002] A prerequisite for a specific and successful therapy is an
exact diagnosis. Specifically in the diagnostic field, the
possibilities have very greatly increased in recent years, whereby,
for example, NMR diagnosis is able to visualize virtually any
anatomical detail selectively and with great accuracy. In many
cases, the corresponding structures are visible only by the
application of contrast media, however. Moreover, the possibility
exists of configuring the contrast media in such a way that they
selectively accumulate in the desired target structures. To this
end, the accuracy of the imaging can be increased with simultaneous
reduction of the required amount of contrast medium.
[0003] As contrast media for NMR diagnosis, chelate complexes of
paramagnetic metals are suitable. The theory and application of
gadolinium(III) chelates as NMR contrast media are explained in
detail in a survey article by P. Caravan et al. in Chem. Rev. 1999,
99, 2293-2352.
[0004] The image intensity in the proton NMR is basically
determined by the water protons. It depends on the nuclear
relaxation times. Complexes of paramagnetic transition metals and
lanthanoids shorten the relaxation times of adjacent protons by
dipolar interactions. The paramagnetic contrast media are not
directly detected, but rather an indirect detection is carried out
based on the fact that the contrast media can change relaxation
times of adjacent protons, such as water protons. Based on their
high magnetic moments and relaxation efficiency, Gd.sup.3+,
Fe.sup.3+ and Mn.sup.2+ are preferred paramagnetic metal cations in
NMR diagnosis.
[0005] An important physical value, which describes the relaxation
behavior of protons, is longitudinal relaxation time T.sub.1.
Tissues with short relaxation times T.sub.1 generally yield images
of higher intensity than those with longer relaxation times. If the
reciprocal value of measured relaxation time T.sub.1 based on
concentration c is applied to a specific paramagnetic ion, straight
lines of rise R are obtained. This rise is also named relaxivity,
which is a measurement of the capacity of the corresponding
paramagnetic ion to shorten the relaxation time of the adjacent
protons.
[0006] The use of radiopharmaceutical agents for diagnostic and
therapeutic purposes has also been known for a long time in the
area of biological and medical research. In particular,
radiopharmaceutical agents are used to visualize specific
structures such as, for example, the skeleton, organs or tissues.
The diagnostic application requires the use of such radioactive
agents, which accumulate after administration specifically in the
structures in patients that are to be examined. These locally
accumulating radioactive agents can then be traced, plotted or
scintigraphed using suitable detectors, such as, for example
scintillation cameras or other suitable recording processes. The
dispersion and relative intensity of the detected radioactive agent
identifies the site of a structure in which the radioactive agent
is found and can visualize the presence of anomalies in structures
and functions, pathological changes, etc.
[0007] Radiopharmaceutical agents can be used in a similar way as
therapeutic agents to irradiate pathological tissues or areas. Such
treatment requires the production of radioactive therapeutic agents
that accumulate in certain structures, organs or tissues.
[0008] Because of their sometimes relatively high toxicity, the
required ions are normally not administered in the form of
water-soluble salts, but rather in the form of chelate complexes.
The latter can be eliminated virtually unchanged from the body. The
smaller the complexes in solution are, the lower is their moment of
inertia and the faster they rotate in solution (Tumbling Motion
Time). The faster a complex rotates, the lower its relaxivity is.
The relaxivity thus increases with the molecular mass of the entire
complex. A high molecular mass can be achieved by binding to
macromolecules. A good NMR contrast medium is distinguished, i.a.,
in that it has a large value for the relaxivity.
[0009] Conjugates of Gd-DTPA (diethylenetriaminepentaacetic acid)
with albumin are described by, for example, M. D. Organ et al. in
Invest. Radiol. 1987, 22, 665-671 and U. Schmiedl et al. in
Radiology 1987, 162, 205-210. Conjugates of macrocyclic metal
complexes and biomolecules are disclosed in WO 95/31444. To improve
the selectivity of contrast media, WO 01/08712 proposes a contrast
medium that comprises at least two metal chelate units as
image-improving groups and at least two "target binding units" for
binding the contrast medium molecule to the desired target molecule
or target organ in the body.
[0010] Large contrast medium molecules with high molar mass are
obtained according to WO 97/02051 by incorporation of macrocyclic
metal complexes in cascade polymers.
[0011] Tetraazacyclododecanetetraacetic acid derivatives of high
stability and good solubility based on deficient charge that are
suitable for binding to biomolecules are described in EP-A-0 565
930.
[0012] The binding of macrocyclic metal complexes to biomolecules
that is described above makes possible both an increase of
relaxivity and selectivity of the contrast medium. The higher the
relaxivity of the contrast medium, the smaller amount of contrast
medium must be administered to the patient and the greater the
opacification in the image. For this reason, it is additionally
desirable to make available NMR contrast media with the highest
possible relaxivity.
[0013] An object of this invention thus consists in making
available improved contrast media for NMR diagnosis and
radiodiagnosis as well as agents for radiotherapy. In particular,
these NMR contrast media are to have as high a relaxivity as
possible and are to accumulate as selectively as possible at a
desired site in the body.
[0014] It has now been found that this object can be achieved,
surprisingly enough, in that a 1,4,7,10-tetraazacyclododecane
macrocyclic compound with special ligands is provided. The new
qualities of the compounds according to the invention are evident
if they are bonded to biomolecules. By the special liganding of the
macrocyclic compound, the relaxivity of the contrast medium that is
obtained is increased, and in addition a fine-tuning of the
relaxivity for a desired use is possible.
[0015] This invention thus relates to compounds of formula I
##STR1## in which [0016] Z represents a hydrogen atom or at least
two Z's represent a metal ion equivalent, [0017] B.sup.1, B.sup.2,
B.sup.3, B.sup.4 are independently selected from the group
consisting of hydrogen atoms and C.sub.1-4-alkyl radicals, [0018]
R.sup.1, R.sup.2, R.sup.3 are independently selected from the group
consisting of hydrogen atoms and straight, branched or cyclic,
saturated or unsaturated C.sub.1-10-alkyl or aryl radicals, which
optionally are substituted with a carboxyl group --SO.sub.3H or
--PO.sub.3H.sub.2, and whereby the alkyl chains of the
C.sub.1-10-alkyl radicals optionally contain an aryl group and/or
1-2 oxygen atoms, provided that at least one of the radicals
B.sup.1, B.sup.2, B.sup.3, B.sup.4, R.sup.1, R.sup.2 and R.sup.3
does not represent a hydrogen atom, [0019] A represents a straight
or branched, saturated or unsaturated C.sub.1-30-hydrocarbon chain
that optionally contains 1-5 oxygen atoms, 1-5 nitrogen atoms
and/or 1-5 --NR' radicals, in which R' is defined as R.sup.1,
R.sup.2 and R.sup.3 but can be selected independently, which
optionally is substituted with 1-3 carboxyl groups, 1-3
--SO.sub.3H, 1-3 --PO.sub.3H.sub.2 and/or 1-3 halogen atoms, in
which optionally 1-3 carbon atoms are present as carbonyl groups,
whereby the chain or a portion of the chain can be arranged
concentrically, and which is configured in such a way that X is
connected via at least 3 atoms to the nitrogen to which A is
bonded, and [0020] X represents a group that can participate in a
reaction with a biomolecule, as well as their salts and their use
for the production of a conjugate with a biomolecule.
[0021] A corresponding macrocyclic compound in which the four
nitrogen atoms of the macrocyclic ring are substituted in each case
with the substituent --CH(CO.sub.2H)CH.sub.2CH.sub.2--CO.sub.2H is
disclosed in P. Caravan et al., Chem. Rev. 1999, 99, 2293-2352. A
possible use of this compound for the production of conjugates with
biomolecules is not disclosed, however. WO 97/02051 discloses
macrocyclic compounds, in which A is a radical
--CH(R.sup.7)--CO--NR.sup.5--U.sup.6, as intermediate compounds for
the production of cascade polymers. EP-A-0-565 930 discloses
macrocyclic compounds, in which A is a radical
--CH(R.sup.6)--C(O)--NH--(CH.sub.2).sub.1-6--NH-D-. An increase in
relaxivity by certain substituents is not disclosed. These
compounds are consequently excluded in the definition of the
compound of formula I in claim 1.
[0022] Unless otherwise indicated, "alkyl radical" is defined here
as a saturated or unsaturated, straight-chain or branched or cyclic
alkyl radical with the indicated number of carbon atoms. If this
radical can contain other groups or atoms, it is understood here
that the other groups or atoms in addition to the already existing
atoms of the radical are present and can be introduced at any
position of the radical including the terminal positions.
[0023] "Aryl" is defined here preferably as phenyl, bisphenyl,
pyridyl, furanyl, pyrrolyl and imidazolyl. Especially preferred is
phenyl.
[0024] "Hydrocarbon chain," which can be arranged completely or
partially concentrically, is defined here preferably as a
hydrocarbon chain such as, for example, an alkyl chain, which can
comprise, for example, an aliphatic or aromatic, optionally
heterocyclic 5- or 6-ring (e.g., phenyl(ene), pyridyl(ene) or
cyclohexyl(ene)) or consists of the latter.
[0025] In the compound of formula I according to the invention,
three of the four nitrogen atoms of the macrocyclic ring are
substituted with optionally substituted acetic acid or carboxylate
methyl radicals. These radicals contribute to the coordination or
to the charge equalization of a coordinated metal ion. Z therefore
stands either for a hydrogen atom or a metal ion equivalent.
[0026] The acetic acid or carboxylate methyl radicals at three of
the nitrogen atoms of the macrocyclic ring in addition can have
substituents R.sup.1, R.sup.2 and R.sup.3. Moreover, the
macrocyclic ring can have substituents B.sup.1, B.sup.2, B.sup.3
and B.sup.4 at four of its carbon atoms. A special feature of the
compounds according to the invention consists in that at least one
of the B.sup.1, B.sup.2, B.sup.3, B.sup.4, R.sup.1, R.sup.2 and
R.sup.3 does not represent a hydrogen atom, i.e., the macrocyclic
ring must have additional substituents either directly on its ring
atoms and/or on the acetic acid or carboxylate methyl substituents
of its nitrogen atoms. By the suitable selection of these
additional substituents, the desired fine-tuning of the relaxivity
of a contrast medium that is produced with use of the compound
according to the invention is carried out.
[0027] B.sup.1, B.sup.2, B.sup.3 and B.sup.4 can be hydrogen atoms
or C.sub.1-4-alkyl radicals. Preferred C.sub.1-4-alkyl radicals are
methyl, ethyl and iso-propyl.
[0028] If B.sup.1, B.sup.2, B.sup.3 and B.sup.4 are hydrogen atoms
in the compounds of formula I according to the invention, R.sup.1,
R.sup.2 and R.sup.3 are independently selected from the group
consisting of hydrogen atoms, straight, branched and/or cyclic,
saturated or unsaturated C.sub.1-10-alkyl (preferably
C.sub.5-10-alkyl) or aryl radicals, which optionally are
substituted with a carboxyl group, --SO.sub.3H or
--PO.sub.3H.sub.2, and whereby the alkyl chains of the
C.sub.1-10-alkyl radicals optionally contain an aryl group and/or
1-2 oxygen atoms, provided that at least one of the radicals
R.sup.1, R.sup.2 and R.sup.3 does not represent a hydrogen atom. As
alkyl radicals, straight-chain or branched, preferably saturated
C.sub.1-10- and especially C.sub.1-4-alkyl radicals, such as
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl and
tert-butyl, as well as cyclohexyl, are preferred. As an
alternative, straight-chain, branched or cyclic, preferably
saturated C.sub.5-10-alkyl radicals, such as pentyl, hexyl,
cyclohexyl, heptyl, octyl, nonyl and decyl, are preferred. The
C.sub.1-10-alkyl radicals for R.sup.1, R.sup.2 and R.sup.3 can
optionally be substituted with a carboxyl group, --SO.sub.3H or
--PO.sub.3H.sub.2. Preferred examples of such substituted alkyl
groups are --CH.sub.2--COOH and --C(CH.sub.3).sub.2--COOH.
Moreover, the alkyl chain of the C.sub.1-10-alkyl radicals can
contain an aryl group and/or 1-2 oxygen atoms. The aryl group and
the oxygen atoms can be present at any position within the alkyl
chain. The aryl group, moreover, can also be arranged in terminal
position on the alkyl chain and can form an aryloxy group together
with an oxygen atom. Especially a phenyl group is suitable as an
aryl group.
[0029] Preferred alkyl chains for R.sup.1, R.sup.2 and R.sup.3,
which optionally contain an aryl group and 1-2 oxygen atoms, are
radicals of formula
--(CH.sub.2).sub.m--(O).sub.n-(phenylene).sub.p-Y, in which m is an
integer from 1-5, n is 0 or 1, p is 0 or 1 and Y is a hydrogen
atom, a methoxy radical, a carboxyl group, --SO.sub.3H or
--PO.sub.3H.sub.2. Substituent Y is preferably in para-position in
this case.
[0030] The aryl radicals for R.sup.1, R.sup.2 and R.sup.3 are
preferably phenyl radicals, which are optionally substituted with a
carboxyl group, --SO.sub.3H or --PO.sub.3H.sub.2.
[0031] If B.sup.1, B.sup.2, B.sup.3 and B.sup.4 are hydrogen atoms,
R.sup.1, R.sup.2 and R.sup.3 are preferably independently selected
from the group consisting of hydrogen atoms, isopropyl, isobutyl,
tert-butyl, a straight-chain or branched C.sub.5-10-alkyl radicals,
cyclohexyl, --CH.sub.2--COOH, --C(CH.sub.3).sub.2--COOH, phenyl
radicals or radicals of formula
--(CH.sub.2).sub.m--(O).sub.n-(phenylene).sub.p-Y, in which m is an
integer from 1 to 5, n is 0 or 1, p is 0 or 1, and Y represents a
hydrogen atom, a methoxy radical, a carboxyl group, --SO.sub.3H or
--PO.sub.3H.sub.2, and R.sup.1, R.sup.2 and R.sup.3 are especially
preferably independently selected from the group consisting of
hydrogen atoms, isopropyl, cyclohexyl or phenyl radicals, provided
that at least one of the radicals R.sup.1, R.sup.2 and R.sup.3 does
not represent a hydrogen atom.
[0032] The substituted macrocyclic ring of the compound of formula
I can be bonded via a spacer A to a biomolecule using a group X,
which can participate in a reaction with a biomolecule.
[0033] In this case, spacer A represents a straight or branched,
saturated or unsaturated C.sub.1-30 hydrocarbon chain, which
optionally contains 1-5 oxygen atoms, 1-5 nitrogen atoms and/or 1-5
--NR' radicals, in which R' is defined as R.sup.1, R.sup.2 and
R.sup.3 above but can be selected independently, which optionally
is substituted with 1-3 carboxyl groups, 1-3 --SO.sub.3H, 1-3
--PO.sub.3H.sub.2 and/or 1-3 halogen atoms, in which optionally 1-3
carbon atoms are present as carbonyl groups, whereby the chain or a
portion of the chain can be arranged concentrically and which is
configured in such a way that X is connected via at least 3 atoms
to the nitrogen atom to which A is bonded.
[0034] The spacer is to have at least three atoms and preferably at
least four atoms in a chain between the nitrogen atom of the
macrocyclic ring and X. A chain of atoms is defined in this case as
the shortest connection between the nitrogen atom of the
macrocyclic ring and X via a ring as well. In terms of this
definition, for example, a para-phenylene group would be regarded
as a spacer with four atoms in a chain, and a meta-phenylene group
would be regarded as a spacer with three atoms in a chain. In
determining the length of the atom chain, carbon, nitrogen and
oxygen atoms are simultaneously counted in each case as an atom.
Substituents in these atoms or side chains are not part of the
number of atoms inside the chain.
[0035] -A-X is preferably selected to be different from the
substituents --CH(R.sup.1)--CO.sub.2Z, --CH(R.sup.2)--CO.sub.2Z and
--CH(R.sup.3)--CO.sub.2Z.
[0036] Spacer A preferably can be represented as a radical A'-U, in
which A' is bonded to the nitrogen atom of the macrocyclic ring and
U is bonded to X. Hereinafter, A' is preferably [0037] a) a bond,
[0038] b) --CH(CO.sub.2H)--, [0039] c) a group of formula ##STR2##
[0040] in which Q represents a hydrogen atom, a C.sub.1-10-alkyl
radical, which optionally is substituted with a carboxyl group, or
Q represents an aryl radical, which optionally is substituted with
a carboxyl group, a C.sub.1-15-alkoxy group, an aryloxy group or a
halogen atom, and R' is defined as R.sup.1, R.sup.2 and R.sup.3,
but can be selected independently, or [0041] d) a group of formula
##STR3## [0042] in which o is 0 or 1, and the ring optionally is
annellated with a benzene ring, whereby this benzene ring, if
present, can be substituted with a methoxy or carboxyl group,
--SO.sub.3H or --PO.sub.3H.sub.2. In the groups above under c) and
d), the positions that are marked ##STR4## [0043] are bonded to the
adjacent groups, position .alpha. is bonded to a nitrogen atom of
the macrocyclic ring, and position .beta. is bonded to U.
[0044] In the group of formula ##STR5## Q is preferably a linear or
branched C.sub.1-10 radical, especially a C.sub.1-4-alkyl radical,
such as methyl, ethyl or isopropyl, or a cyclohexyl radical. These
radicals can optionally be substituted with a carboxyl group,
whereby a carboxymethyl radical is preferred. The preferred aryl
radical for Q is phenyl. This aryl radical can be substituted with
a carboxyl group, a C.sub.1-15-alkoxy group, an aryloxy group, such
as especially a phenoxy group, or a halogen atom, such as fluorine,
chlorine, bromine or iodine, and especially fluorine or chlorine.
If the aryl radical is a phenyl radical, the latter is preferably
substituted in para-position with one of the above-mentioned
groups. Especially preferred groups for Q are methyl, phenyl and
p-dodecanoxyphenyl.
[0045] R'is defined as R.sup.1, R.sup.2 and R.sup.3 above, but can
be selected independently from R.sup.1, R.sup.2 and R.sup.3. R' is
especially preferably a hydrogen atom.
[0046] A' is preferably selected from a bond, --CH(CO.sub.2H)--,
--C(CH.sub.3)H--CO--NH--, --C(phenyl)H--CO--NH--,
--C(p-dodecanoxyphenyl)H--CO--NH--, ##STR6## in which R.sup.4 is
--OCH.sub.3, --CO.sub.2H, --SO.sub.3H or --PO.sub.3H.sub.2.
[0047] If spacer A is represented as a radical A'-U, and A' has the
meaning defined above, U is preferably a straight or branched,
saturated or unsaturated C.sub.1-30-hydrocarbon chain, which
optionally contains 1-3 oxygen atoms, 1-3 nitrogen atoms and/or 1-3
--NR'' radicals, in which R'' is defined as R.sup.1, R.sup.2 and
R.sup.3 above, but can be selected independently, and in which
optionally 1-3 carbon atoms are present as carbonyl groups, whereby
the chain or a portion of the chain can be arranged concentrically.
U is especially preferably an aryl radical or a C.sub.1-20-alkyl
radical (preferably straight-lined or at least partially cyclic and
saturated) that optionally contains 1-3 oxygen atoms, 1-3 NR''
radicals, 1-2 phenylene radicals and/or a pyridylene radical, in
which optionally 1-3 carbon atoms are present as carbonyl groups,
and which optionally is substituted with an aryl radical (e.g.,
phenyl). A' and U together must be configured in such a way that X
is connected by at least three atoms to the nitrogen atom to which
A' is bonded. The chain of at least three atoms is defined as above
in A.
[0048] The aryl radical for U is preferably a phenyl radical. The
C.sub.1-20-alkyl radical for U is preferably a linear, saturated
C.sub.1-10-alkyl radical, cyclohexyl radical or
cyclohexyl-C.sub.1-5-alkyl radical. The alkyl radicals of these
radicals can optionally be interrupted by 1 oxygen atom, 1
phenylene radical and/or 1 pyridylene radical or can contain a
--CO--NR'' radical or can be substituted with phenyl. U is
preferably selected from --CH.sub.2--, --(CH.sub.2).sub.5--,
--(CH.sub.2).sub.10--, -phenylene-O--CH.sub.2--,
-phenylene-O--(CH.sub.2).sub.3--,
-phenylene-O--(CH.sub.2).sub.10--, --CH.sub.2-phenylene-,
-cyclohexylene-O--CH.sub.2--, -phenylene-, --C(phenyl)H--,
--CH.sub.2-pyridylene-O--CH.sub.2--, --CH.sub.2-pyridylene- and
--CH.sub.2--CO--NH--CH.sub.2--CH.sub.2--. In the above-mentioned
preferred groups for U, the phenylene groups are preferably
substituted in para-position, and the pyridylene groups are
preferably pyrid-2,5-ylene groups or pyrid-2,4-ylene groups.
[0049] Preferred groups for the spacer A are: ##STR7## ##STR8##
[0050] Via spacer A, a group X is bonded to the macrocyclic ring in
the compounds of formula I. This group X is a group that can
participate in a reaction with a biomolecule. For this purpose, for
example, carboxyl (--COOH), activated carboxyl, amino (--NH.sub.2),
isocyanate (--NCO), isothiocyanate (--NCS), hydrazine
(--NHNH.sub.2), semicarbazide (--NHCONHNH.sub.2), thiosemicarbazide
(--NHCSNHNH.sub.2), chloroacetamide (--NHCOCH.sub.2Cl),
bromoacetamide (--NHCOCH.sub.2Br), iodoacetamide (--NHCOCH.sub.2I),
acylamino, such as, for example acetylamino (--NHCOCH.sub.3), mixed
anhydrides, azide, hydroxide, sulfonyl chloride, carbodiimide or a
group of formulas ##STR9## in which Hal represents a halogen atom,
is suitable.
[0051] Activated carboxyl groups are defined above as those
carboxyl groups that can be derivatized in such a way that they
facilitate the reaction with a biomolecule. Which groups can be
used for activation is known, and reference can be made to, for
example, M. and A. Bodanszky, "The Practice of Peptide Synthesis,"
Springerverlag 1984. Examples are aducts of carboxylic acid with
carbodiimides or activated esters, such as, e.g.,
hydroxybenzotriazole esters. Especially preferred is the activated
carboxyl group for X that is selected from ##STR10##
[0052] In formula I, Z stands for a hydrogen atom or a metal ion
equivalent. Which metal ion in the compound according to the
invention is to be complexed here depends on the intended use of
the conjugates that are produced with the inventions according to
the invention with a biomolecule. Corresponding conjugates are
suitable, for example, for NMR diagnosis, radiodiagnosis and
radiotherapy and neutron capture therapy. The conjugates in NMR
diagnosis are especially preferably used as contrast media.
[0053] The production of complexes for NMR diagnosis can be carried
out as was disclosed in Patents EP 71564, EP 130934 and DE-OS 34 01
052. To this end, the metal oxide or a metal salt (for example a
chloride, nitrate, acetate, carbonate or sulfate) of the desired
element is dissolved or suspended in water and/or a lower alcohol
(such as methanol, ethanol or isopropanol) and reacted with the
solution or suspension of the equivalent amount of the complexing
agent according to the invention.
[0054] If the complexing agents are to be used for the production
of radiodiagnostic agents or radiotherapeutic agents, the
production of the complexes from the complexing agents can be
carried out according to the methods that are described in
"Radiotracers for Medical Applications," Vol. I, CRC Press, Boca
Raton, Fla.
[0055] The compounds according to the invention are used [0056] 1.
For NMR diagnosis in the form of their complexes with the ions of
the paramagnetic elements with atomic numbers 21-29, 42, 44 and
58-70.
[0057] Suitable ions are, for example, the chromium(III), ion(II),
cobalt(II), nickel(II), copper(II), praseodymium(III),
neodymium(III), samarium(III) and ytterbium(III) ion. Because of
their strong magnetic moment, the gadolinum(II), terbium(III),
dysprosium(III), holmium(III), erbium(III), manganese(II) and
iron(III) ions are especially preferred for NMR diagnosis. [0058]
2. For radiodiagnosis and radiotherapy in the form of their
complexes with the radioisotopes of elements with atomic numbers
26, 27, 29, 31, 32, 37-39, 43, 46, 47, 49, 61, 62, 64, 67, 70, 71,
75, 77, 82 and 83.
[0059] The compounds according to the invention and especially
their conjugates with biomolecules meet the many different
requirements for suitability as contrast media for nuclear spin
tomography. After oral or parenteral administration, they are thus
extremely well suited for enhancing the informational value of the
image that is obtained with the aid of a nuclear spin tomograph by
increasing the signal intensity. They also show the high
effectiveness that is necessary to load the body with the smallest
possible amounts of foreign substances and the good compatibility
that is necessary to maintain the non-invasive nature of the
studies.
[0060] The good water solubility and low osmolality of the
compounds according to the invention and their conjugates with
biomolecules allow for the production of highly concentrated
solutions so as to keep the volume burden of the circulatory system
within reasonable limits and to offset the dilution by bodily
fluids, i.e., NMR diagnostic agents have to be 100 to 1000 times
more water-soluble than for NMR spectroscopy. In addition, the
compounds according to the invention have not only a high stability
in vitro but also a surprisingly high stability in vivo, so that a
release or an exchange of the ions, which are inherently toxic and
not covalently bonded in the complexes, is carried out only
extremely slowly within the time that it takes for the new contrast
media to be completely excreted again.
[0061] The complex compounds according to the invention can also be
used advantageously as susceptibility reagents and as shift
reagents for in vivo NMR spectroscopy.
[0062] The compounds according to the invention and their
conjugates with biomolecules are also suitable as radiodiagnostic
agents and radiotherapeutic agents based on their advantageous
radioactive properties and the good stability of the complex
compounds that are contained therein. Details of their use and
dosage are described in, e.g., "Radiotracers for Medical
Applications," CRC Press, Boca Raton, Fla. 1983, as well as in Eur.
J. Nucl. Med. 17 (1990) 346-364 and Chem. Rev. 93 (1993)
1137-1156.
[0063] For SPECT, the complexes with isotopes .sup.111In and
.sup.99mTc are suitable.
[0064] Another imaging method with radioisotopes is the
positron-emission tomography, which uses positron-emitting isotopes
such as, e.g., .sup.43Sc, .sup.44Sc, .sup.52Fe, .sup.55Co,
.sup.68Ga, .sup.64Cu, .sup.86Y and .sup.94mTc (Heiss, W. D.;
Phelps, M. E.; Positron Emission Tomography of Brain, Springer
Verlag Berlin, Heidelberg, New York 1983).
[0065] The compounds according to the invention and their
conjugates with biomolecules are also suitable, surprisingly
enough, for differentiating malignant and benign tumors in areas
without blood-brain barriers.
[0066] They are distinguished in that they are completely
eliminated from the body and thus are well-tolerated.
[0067] Since the compounds according to the invention and
especially their conjugates with biomolecules accumulate in
malignant tumors (no diffusion in healthy tissue, but high
permeability of tumor vessels), they can also support the radiation
therapy of malignant tumors. The latter is distinguished from the
corresponding diagnosis only by the amount and type of the isotope
that is used. The purpose in this case is the destruction of tumor
cells by high-energy short-wave radiation with the lowest possible
range of action. For this purpose, interactions of the metals that
are contained in the complexes (such as, e.g., iron or gadolinium)
with ionizing radiations (e.g., x rays) or with neutron rays are
employed. By this effect, the local radiation dose at the site
where the metal complex is found (e.g., in tumors) increases
significantly. To produce the same radiation dose in the malignant
tissue, radiation exposure for healthy tissue can be considerably
reduced and thus burdensome side effects for the patients can be
avoided when such metal complexes are used. The metal complex
conjugates according to the invention are therefore also suitable
as radio-sensitizing substances in the radiation therapy of
malignant tumors (e.g., exploiting Mossbauer effects or neutron
capture therapy). Suitable .beta.-emitting ions are, e.g.,
.sup.46Sc, .sup.47Sc, .sup.48Sc, .sup.72Ga, .sup.73Ga, .sup.90Y,
.sup.67Cu, .sup.109Pd, .sup.111Ag, .sup.149Pm, .sup.153Sm,
.sup.166Ho, .sup.177Lu, .sup.186 Re and .sup.188Re .sup.90Y,
.sup.177Lu, .sup.72Ga, .sup.153Sm and .sup.67Cu are preferred.
Suitable .alpha.-emitting ions that have short half-lives are,
e.g., .sup.211At, .sup.211Bi, .sup.212Bi, .sup.213Bi and
.sup.214Bi, whereby .sup.212Bi is preferred. A suitable photon- and
electron-emitting ion is .sup.158Gd, which can be obtained from
.sup.157Gd by neutron capture.
[0068] If the compound according to the invention or conjugate
thereof with a biomolecule is intended for use in the variant of
the radiation therapy that is proposed by R. L. Mills et al.
[Nature Vol. 336 (1988), p. 787], the central ion must be derived
from a Mossbauer isotope, such as, for example, .sup.57Fe or
.sup.151Eu.
[0069] The neutralization of optionally still present free carboxy
groups is carried out with the aid of inorganic bases (e.g.,
hydroxides, carbonates or bicarbonates) of, e.g., sodium,
potassium, lithium, magnesium or calcium and/or organic bases, such
as, i.a., primary, secondary and tertiary amines, such as, e.g.,
ethanolamine, morpholine, glucamine, N-methylglucamine and
N,N-dimethylglucamine, as well as basic amino acids, such as, e.g.,
lysine, arginine and ornithine or amides of originally neutral or
acidic amino acids.
[0070] For the production of natural complex compounds, as much of
the desired base can be added, for example, into acid complex salts
in aqueous solution or suspension so that the neutral point is
reached. The solution that is obtained can then be evaporated to
the dry state in a vacuum. It is often advantageous to precipitate
the neutral salts that are formed by adding water-miscible
solvents, such as, e.g., lower alcohols (methanol, ethanol,
isopropanol, etc.), lower ketones (acetone, etc.), polar ethers
(tetrahydrofuran, dioxane, 1,2-dimethoyethane, etc.) and thus to
obtain easily isolated and readily purified crystallizates. It has
proven especially advantageous to add the desired base as early as
during the complexing of the reaction mixture and thus to save a
process step.
[0071] The compounds of formula I according to the invention can be
produced according to the process that is known to one skilled in
the art. For example, the compounds of formula I can be obtained by
a process in which a compound of formula II ##STR11## in which
B.sup.1, B.sup.2, B.sup.3 and B.sup.4 are defined as above is
optionally reacted after introducing protective groups for the
nitrogen atoms with Nu-A-X' and Nu-CH(R.sup.1)--CO.sub.2Z',
Nu-CH(R.sup.2)--CO.sub.2Z' and Nu-CH(R.sup.3)--CO.sub.2Z', whereby
A and R.sup.1, R.sup.2, and R.sup.3 are defined as above and Nu is
a nucleofuge, X' stands for X or a protected form of X, and X is
defined as above and Z' stands for a hydrogen atom, a metal ion
equivalent, preferably an alkali metal or alkaline-earth metal,
such as especially sodium or potassium, or a protective group for
carboxyl. Then, the optionally present protective groups can be
removed, and it can be reacted in a way that is known in the art
with at least one metal oxide or metal salt of a desired element.
Then, in the thus obtained complexes, still present acid hydrogen
atoms optionally can be substituted completely or partially by
cations of inorganic and/or organic bases, amino acids or amino
acid amides.
[0072] Three preferred process variants are described in more
detail below.
Further process variants, especially where R.sup.1, R.sup.2 and
R.sup.3 do not represent the same radicals, are shown in the
examples. The described process variants do not limit the extend of
the invention.
[0073] In the compounds according to the invention, B.sup.1,
B.sup.2, B.sup.3 and B.sup.4 can represent the same residues,
whereas R.sup.1, R.sup.2, and/or R.sup.3 can represent different
residues. Furthermore B.sup.1, B.sup.2, B.sup.3 and B.sup.4 can
represent different residues in the molecule and R.sup.1, R.sup.2,
and/or R.sup.3 can represent the same residues. And in the
compounds according to the invention B.sup.1, B.sup.2, B.sup.3 and
B.sup.4 can represent different residues and also R.sup.1, R.sup.2,
and/or R.sup.3 can represent different residues. Furthermore
B.sup.1, B.sup.2, B.sup.3 and B.sup.4 can represent the same
residues in the molecule and R.sup.1, R.sup.2, and/or R.sup.3 can
also represent the same residues.
[0074] In the first variant, the macrocyclic compound that is
unsubstituted at the nitrogens is first reacted with protected unit
AX'. In this case, group A carries a nucleofuge as a leaving group.
By stoichiometric reaction control, one of the four nitrogen atoms
in the macrocyclic compound reacts with group A with the leaving
group departing. In this way, a monofunctionalized macrocyclic
compound that contains radical X in protected form (X') is
obtained. In the second reaction step, the remaining three
nucleophilic nitrogen atoms of the macrocyclic compound are reacted
in each case with a protected carboxylic acid, which carries a
nucleofuge in .alpha.-position in the carboxyl group.
[0075] For example three equivalents of the protected carboxylic
acids can be used in the second reaction step. It is possible to
use one protected carboxylic acid. In this case, all residues
R.sup.1R.sup.2 and R.sup.3 are the same. Alternatively up to three
different protected carboxylic acids can be used in same process.
In this case it is possible to introduce different residues
R.sup.1, R.sup.2 and/or R.sup.3 into the molecule, wherein R.sup.1,
R.sup.2 and R.sup.3 are as defined above. Of course the different
protected carboxylic acids should add up to about 3 equivalents as
a total. The different protected carboxylic acids can be used as a
mixture, but it is preferred to add the different protected
carboxylic acids in different reaction steps. With this method it
is possible to obtain a well defined chemical compound or a mixture
of chemical compounds having different substitution patterns. If a
mixture of chemical compounds with different substitution patterns
is obtained, this mixture can be seperated by known methods, such
as chromatography, after the reaction. Suitable synthetic
approaches for obtaining chemical compounds, in which R.sup.1,
R.sup.2 and R.sup.3 are different are shown in the examples and
these exemplified processes can easily be adapted by a skilled
person to obtain further compounds having different residues
R.sup.1, R.sup.2 and/or R.sup.3. These and further synthetic
approaches are known in the art and can employ steps of e.g.
addition or cleavage of protecting groups in order to selectively
choose the substituents for each N-atom independently. After the
protective groups are cleaved off from the carboxylic acid
functionalities, the complex that consists of paramagnetic metal
ions and chelate ligands is finished by adding metal oxide or metal
salt. This process variant is diagrammatically reproduced below,
whereby the radicals in the formulas are defined as above:
##STR12##
[0076] In a second variant, a macrocyclic compound is used as an
educt, which carries already suitable protective groups SG on three
of the four nitrogen atoms. As protective groups, e.g.,
tert-butyl-oxycarbonyl (t-BOC), COCF.sub.3, carbobenzoxy (Cbo) or
fluorenyl-methoxycarbonyl (FMOC), etc. are suitable here. By the
presence of the protective groups, only one of the four nitrogen
atoms is nucleophilic and can react with A-X', which for its part
carries a nucleofuge Nu as in the variant above. After linkage of
both molecules with the leaving group departing, a cleavage of the
three protective groups from the nitrogen atoms is carried out. It
follows the derivatization with the aid of the carboxylic acid
derivatives, as was already described for the variants above. This
second process variant is diagrammatically reproduced below,
whereby the radicals in the formulas are defined as above:
##STR13##
[0077] SG=Protective group (e.g., BOC, Cbo, COCF.sub.3, FMOC,
etc.)
[0078] In the third variant, first one of the four nitrogen atoms
of the macrocyclic compound is blocked by a corresponding
protective group SG. Examples of suitable protective groups are
formyl, benzyl, boctrityl, etc. The reaction now is carried out on
the three remaining nucleophilic nitrogen atoms with
correspondingly protected carboxylic acid derivatives, which carry
a corresponding nucleofuge in .alpha.-position. For example three
equivalents of the protected carboxylic acids can be used in the
second reaction step. It is possible to use one protected
carboxylic acid. In this case, all residues R.sup.1, R.sup.2 and
R.sup.3 are the same. Alternatively up to three different protected
carboxylic acids can be used in same process. In this case it is
possible to introduce different residues R.sup.1, R.sup.2 and/or
R.sup.3 into the molecule, wherein R.sup.1, R.sup.2 and R.sup.3 are
as defined above. Of course the different protected carboxylic
acids should add up to about 3 equivalents as a total. The
different protected carboxylic acids can be used as a mixture, but
it is preferred to add the different protected carboxylic acids in
different reaction steps. With this method it is possible to obtain
a well defined chemical compound or a mixture of chemical compounds
having different substitution patterns. If a mixture of chemical
compounds with different substitution patterns is obtained, this
mixture can be seperated by known methods, such as chromatography,
after the reaction. Suitable synthetic approaches for obtaining
chemical compounds, in which R.sup.1, R.sup.2 and R.sup.3 are
different are shown in the examples and these exemplified processes
can easily be adapted by a skilled person to obtain further
compounds having different residues R.sup.1, R.sup.2 and/or
R.sup.3. These and further synthetic approaches are known in the
art and can employ steps of e.g. addition or cleavage of protecting
groups in order to selectively choose the substituents for each
N-atom independently. Then, the cleavage of protective group SG
that is first introduced at the first nitrogen atom and
derivatizing with AX', which for its part also carries a
nucleofuge, are carried out. This third process variant is
diagrammatically reproduced below, whereby the radicals in the
formulas are defined as above: ##STR14##
[0079] Advantageously used as a nucleofuge are the radicals:
[0080] Cl, Br, I, O-triflate, mesylate and tosylate.
[0081] The reaction is performed in a mixture of water and organic
solvents, such as: isopropanol, ethanol, methanol, butanol,
dioxane, tetrahydrofuran, dimethylformamide, dimethyl acetamide,
formamide or dichloromethane. Ternary mixtures that consist of
water, isopropanol and dichloromethane are preferred.
[0082] The reaction is carried out in a temperature range of
between -10.degree. C. and 100.degree. C., preferably between
0.degree. C. and 30.degree. C.
[0083] The protection of the above-named groups can be accomplished
in numerous ways that are familiar to one skilled in the art. The
embodiments that are described below are used to explain these
protective group techniques without being limited to these
synthesis methods.
[0084] As acid protective groups, C.sub.1-C.sub.6-alkyl,
C.sub.6-C.sub.10-aryl and
C.sub.6-C.sub.10--Ar(C.sub.1-C.sub.4)-alkyl groups as well as
trialkylsilyl groups are suitable. The methyl, ethyl, propyl,
isopropyl, n-butyl, i-butyl and tert-butyl groups are
preferred.
[0085] The cleavage of these acid protective groups is carried out
according to the processes that are known to one skilled in the
art, for example by hydrolysis, hydrogenolysis, alkaline
saponification of the esters with alkali in aqueous-alcoholic
solution at temperatures from 0 to 50.degree. C., acidic
saponification with mineral acids or in the case of tert-butyl
esters with the aid of trifluoroacetic acid.
[0086] The NH groups can be protected in a variety of ways and then
exposed again. The N-trifluoroacetyl derivative is cleaved by
potassium or sodium carbonate in water (H. Newman, J. Org. Chem.,
30: 287 (1965), M. A. Schwartz et al., J. Am. Chem. Soc., 95 G12
(1973)) or simply by ammonia solution (M. Imazama and F. Eckstein,
J. Org. Chem., 44: 2039 (1979)). The tert-butyloxycarbonyl
derivative is equally easy to cleave: stirring with trifluoroacetic
acid suffices (B. F. Lundt et al., J. Org. Chem., 43: 2285 (1978)).
The group of NH protective groups to be cleaved hydrogenolytically
or in a reductive manner is very large: the N-benzyl group can be
cleaved easily with hydrogen/Pd--C (W. H. Hartung and R. Rimonoff,
Org. Reactions VII, 262 (1953)), which also applies for the trityl
group (L. Zervas et al., J. Am. Chem. Soc., 78; 1359 (1956)) and
the benzyloxycarbonyl group (M. Bergmann and L. Zervas Ber. 65:
1192 (1932)).
[0087] The activated esters of the above-described compounds are
produced as known to one skilled in the art. For the case of
isothiocyanates or .alpha.-haloacetates, the corresponding terminal
amino precursors are reacted according to methods that are known in
the literature with thiophosgene or 2-halo-acetic acid-halides. The
reaction with correspondingly derivatized esters of
N-hydroxysuccinimide, such as, for example: ##STR15## is also
possible (Hal=halogen).
[0088] In general, for this purpose, all commonly used activation
methods for carboxylic acids that are known in the prior art can be
used. The molecule Nu-A-X is preferably synthesized first
independently. If the molecule contains an amide group, the latter
is produced, for example, by an activated carboxylic acid being
reacted with an amine. The activation of the carboxylic acid is
carried out according to the commonly used methods. Examples of
suitable activating reagents are dicyclohexylcarbodiimide (DCC),
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-hydrochloride (EDC),
benzotriazol-1-yloxytris-(dimethylamino)-phosphonium
hexafluorophosphate (BOP) and
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosp-
hate (HBTU), preferably DCC. The addition of O-nucleophilic
catalysts, such as, e.g., N-hydroxysuccinimide (NHS) or
N-hydroxybenzotriazole, is also possible.
[0089] If group X is a carboxylic acid function, the latter can be
used in protected form (e.g., in the form of benzyl ester), and the
cleavage of the protective group can then be carried out
hydrogenolytically.
[0090] To link this carboxylic acid function to a suitable
functional group of a suitable biomolecule, the latter should
normally first be activated. Esters that are activated to this end
are preferably produced at an intermediate stage, and said esters
are then attacked by a nucleophilic group of the biomolecule. In
this way, a covalent linkage between the biomolecule and the
compound of formula I according to the invention is produced.
Preferred activated esters are the esters of the
N-hydroxysuccinimide, the esters of paranitrophenol or the esters
of pentafluorophenol. If group X in the form of an isothiocyanate
is linked to the biomolecule, a terminal amine is preferably first
used which, if necessary, can be provided with a suitable
protective group. Suitable protective groups are known from peptide
chemistry. After the protective group is cleaved off, the
isothiocyanate can be produced by reaction of the primary terminal
amine with thiophosgene. Nucleophilic groups of the biomolecule can
be added to the latter.
[0091] In an embodiment, group X represents a maleinimide, which
can react, e.g., selectively with thiol functions of the
biomolecule.
[0092] In another embodiment, group X is a nucleophile (NH.sub.2,
SH), which affects a suitable functionality of the biomolecule
(activated ester, maleinimide, etc.). Numerous biomolecules that
are functionalized with maleinimides are commercially
available.
[0093] This invention, moreover, relates to the use of the
above-described compounds of formula I for the production of
conjugates with a biomolecule.
[0094] The synthesis of the conjugates is generally carried out in
such a way that first a derivatized and functionalized chelate
complex is produced that then is linked to the biomolecule. It is
also possible, however, that if synthetically produced biomolecules
are used, the chelate complex according to the invention is
incorporated in the latter during the synthesis of the biomolecule.
This can be carried out, for example, during the sequential
synthesis of oligopeptides in the synthesizing robot. If necessary,
the protective groups that are commonly used in the synthesis of
the corresponding biomolecule can be introduced into the compound
according to the invention. The latter are then cleaved again in
the synthesizer in line with the usual synthesis algorithm.
[0095] "Biomolecule" is defined here as any molecule that either
occurred naturally, for example in the body, or was produced
synthetically with an analogous structure. Moreover, among the
latter, those molecules are defined that can occur in interaction
with a biological molecule that occurs, for example, in the body or
a structure that occurs there, in such a way, for example, that the
conjugates accumulate at specific desired spots of the body. "Body"
is defined here as any plant or animal body, whereby animal and
especially human bodies are preferred.
[0096] Biomolecules are especially the molecules that occur in
living creatures that as products of an evolutionary selection by
orderly and complex interactions meet specific objects of the
organism and constitute the basis of its vital functions (changes
in material and shape, reproduction, energy balance). In
biomolecules, simple building blocks (amino acids, nucleobases,
monosaccharides, fatty acids, etc.) of large molecules (proteins,
nucleic acids, polysaccharides, lipids, etc.) are used in most
cases. Corresponding macromolecules are also referred to as
biopolymers.
[0097] The biomolecule advantageously can have, for example, a
polypeptide skeleton that consists of amino acids with side chains
that can participate in a reaction with reactive group X of the
compounds of formula I according to the invention. Such side chains
include, for example, the carboxyl groups of aspartic acid and
glutamic acid esters, the amino groups of lysine radicals, the
aromatic groups of tyrosine and histidine radicals and the
sulfhydryl groups of cysteine radicals.
[0098] A survey on biomolecules with numerous examples is found in
the manuscript "Chemie der Biomolekule [Chemistry of Biomolecules]"
of TU-Graz (H. Berthold et al., Institut fur Organische Chemie
[Institute for Organic Chemistry], Tu-Graz, 2001), which can also
be seen on the Internet under www.orgc.tu-graz.ac.at. The content
of this document is integrated by reference in this
description.
[0099] To form conjugates with the compounds according to the
invention, the following biomolecules are especially suitable:
[0100] Biopolymers, proteins, such as proteins that have a
biological function, HSA, BSA, etc., proteins and peptides, which
accumulate at certain spots in the organism (e.g., in receptors,
cell membranes, at ducts, etc.), peptides that can be cleaved by
proteases, peptides with predetermined synthetic sites of rupture
(e.g., labile esters, amides, etc.), peptides that are cleaved by
metalloproteases, peptides with photocleavable linkers, peptides
with oxidative agents (oxydases) and cleavable groups, peptides
with natural and unnatural amino acids, glycoproteins
(glycopeptides), signal proteins, antiviral proteins and apoctosis,
synthetically modified biopolymers such as biopolymers that are
derivatized with linkers, modified metalloproteases and derivatized
oxydase, etc., carbohydrates (mono- to polysaccharides), such as
derivatized sugars, sugars that can be cleaved in the organism,
cyclodextrins and derivatives thereof, amino sugars, chitosan,
polysulfates and acetylneuraminic acid derivatives, antibodies,
such as monoclonal antibodies, antibody fragments, polyclonal
antibodies, minibodies, single chains (also those that are linked
by linkers to multiple fragments), red blood corpuscles and other
blood components, cancer markers (e.g., CAA) and cell adhesion
substances (e.g., Lewis X and anti-Lewis X derivatives), DNA and
RNA fragments, such as derivatized DNAs and RNAs (e.g., those that
were found by the SELEX process), synthetic RNA and DNA (also with
unnatural bases), PNAs (Hoechst) and antisense, .beta.-amino acids
(Seebach), vector amines for transfer into the cell, biogenic
amines, pharmaceutical agents, oncological preparations, synthetic
polymers, which are directed to a biological target (e.g.,
receptor), steroids (natural and modified), prostaglandins, taxol
and derivatives thereof, endothelins, alkaloids, folic acid and
derivatives thereof, bioactive lipids, fats, fatty acid esters,
synthetically modified mono-, di- and triglycerides, liposomes,
which are derivatized on the surface, micelles that consist of
natural fatty acids or perfluoroalkyl compounds, porphyrins,
texaphrines, expanded porphyrins, cytochromes, inhibitors,
neuramidases, neuropeptides, immunomodulators, such as FK 506, CAPE
and gliotoxin, endoglycosidases, substrates that are activated by
enzymes such as calmodulin kinase, casein-kinase II,
glutathione-S-transferase, heparinase, matrix-metalloproteases,
.beta.-insulin-receptor-kinase, UDP-galactose 4-epimerase,
fucosidases, G-proteins, galactosidases, glycosidases,
glycosyltransferases and xylosidase, antibiotics, vitamins and
vitamin analogs, hormones, DNA intercalators, nucleosides,
nucleotides, lectins, vitamin B12, Lewis-X and related substances,
psoralens, dienetriene antibiotics, carbacyclins, VEGF (vascular
endothelial growth factor), somatostatin and derivatives thereof,
biotin derivatives, antihormones, tumor-specific proteins and
synthetic agents, polymers that accumulate in acidic or basic areas
of the body (pH-controlled dispersion), myoglobins, apomyoglobins,
etc., neurotransmitter peptides, tumor necrosis factors, peptides
that accumulate in inflamed tissues, blood-pool reagents, anion and
cation-transporter proteins, polyesters (e.g., lactic acid),
polyamides and polyphosphates.
[0101] Most of the above-mentioned biomolecules are commercially
available from, for example, Merck, Aldrich, Sigma, Calibochem or
Bachem.
[0102] In addition, all "plasma protein binding groups" or "target
binding groups" that are disclosed in WO 96/23526 and WO 01/08712
can be used as biomolecules. The content of these two laid-open
specifications is therefore integrated by reference into this
description.
[0103] The number of compounds of formula I according to the
invention per biomolecule is random in principle, but a molecular
ratio of 0.1:1 to 10:1, especially 0.5:1 to 7:1, is preferred.
[0104] The compounds according to the invention are also suitable
for conjugation on all molecules that are reacted with fluorescence
dyes in the prior art to determine, for example, their location by
epifluorescence microscopy within the cell. After the
administration of the medication, the compounds with, in principle,
any medications can also be conjugated to then track the transport
within the organism, for example by the NMR technique. It is also
possible that the conjugates from the compounds according to the
invention and the biomolecules contain other additional molecules,
which had been conjugated on the biomolecules. The term
"biomolecule" in terms of this invention thus encompasses all
molecules that occur in the biological systems and all molecules
that are biocompatible.
[0105] The conjugates that are obtained with the compounds
according to the invention are preferably used as contrast media in
NMR diagnosis. The conjugates should therefore be water-soluble. If
the conjugates that are obtained with the compounds according to
the invention are to be used as NMR contrast media, they are
preferably dosed in an amount of 0.0001-5 mmol/kg of body weight
and especially preferably in an amount of 0.005-0.5 mmol/kg of body
weight. Details of use are discussed in, e.g., H.-J. Weinmann et
al., Am. J. of Roentgenology 142, 619 (1984). By the surprisingly
high relaxivity of the compounds according to the invention with
simultaneous target specificity of the conjugates that are obtained
with these compounds, the latter can be especially low-dosed, for
example, to detect tumors.
[0106] Details of use of radiotherapeutic agents are discussed in,
e.g., R. W. Kozak et al. TIBTEC, October 1986, 262 (see above
Bioconjugate Chem. 12 (2001) 7-34).
[0107] This invention is explained in more detail by the examples
below without being limited thereto.
[0108] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever.
[0109] In the foregoing and in the following examples, all
temperatures are set forth uncorrected in degrees Celsius and, all
parts and percentages are by weight, unless otherwise
indicated.
EXAMPLES
Example 1
a)
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7-.alpha.,.alp-
ha.',.alpha.''-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tet-
raazacyclododecane
[0110] 25 g (81.1 mmol) of 2-bromopropionylglycine-benzyl ester
(Example 1e of WO 98/24774) is added to 27.9 g (162.2 mol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained
1-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacycl-
ododecane (19.6 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) of
N-ethyldiisopropylamine in 200 ml of dichloromethane are added to
62.45 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid
benzyl ester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3),
360) in 400 ml of dichloromethane, and it is stirred for 6 hours
under reflux and then overnight at room temperature. It is
extracted three times with 500 ml of water each, the organic phase
is dried on magnesium sulfate and evaporated to the dry state. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0111] Yield: 32.0 g (73% of theory) of a colorless, crystalline
powder
[0112] Elementary analysis:
[0113] Cld.: C, 68.39; H, 7.23; N, 7.98.
[0114] Fnd.: C, 67.95; H, 7.41; N, 8.22.
b)
10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.-
''-trimethyl-1,4,7-tris(carboxy-methyl)-1,4,7,10-tetraazacyclododecane
[0115] 26.3 g (30 mmol) of the title compound of Example 1a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0116] Yield: 15.7 g (quantitative) of a colorless powder
[0117] Elementary analysis:
[0118] Cld.: C, 51.05; H, 7.60; N, 13.53.
[0119] Fnd.: C, 50.71; H, 7.83; N, 13.25.
c) Gd Complex of
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-
-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0120] 10.4 g (20 mmol) of the ligand that is described in Example
1b is dissolved in 200 ml of water and 80 ml of isopropanol, and it
is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20:1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0121] Yield: 10.1 g (69% of theory) of a colorless powder.
[0122] Water content (Karl-Fischer): 8.3%
[0123] Elementary analysis (relative to anhydrous substance):
[0124] Cld.: C, 39.33; H, 5.40; Gd, 23.41; N, 10.42.
[0125] Fnd.: C, 39.21; H, 5.88; Gd, 22.93; N, 10.11.
Example 2
a)
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7-.alpha.,.alp-
ha.',
.alpha.''-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,-
10-tetraazacyclododecane
[0126] 19.6 g (50 mmol) of the
1-[4-(benzyloxy-carbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyc-
lododecane that is described in Example 1a as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 68.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-isovaleric acid benzyl ester
(Walker et al., Tetrahedron (1997), 53(43), 14591) in 400 ml of
dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0127] Yield: 33.7 g (70% of theory) of a colorless, crystalline
powder
[0128] Elementary analysis:
[0129] Cld.: C, 69.90; H, 7.86; N, 7.28.
[0130] Fnd.: C, 69.77; H, 7.51; N, 7.22.
b)
10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.-
''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-
e
[0131] 28.9 g (30 mmol) of the title compound of Example 2a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0132] Yield: 18.0 g (quantitative) of a colorless powder
[0133] Elementary analysis:
[0134] Cld.: C, 55.89; H, 8.54; N, 11.64.
[0135] Fnd.: C, 55.63; H, 8.83; N, 11.31.
c) Gd Complex of
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-
-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0136] 12.0 g (20 mmol) of the ligand that is described in Example
2b is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0137] Yield: 12.0 g (72% of theory) of a colorless powder.
[0138] Water content (Karl-Fischer): 9.1%
[0139] Elementary analysis (relative to anhydrous substance):
[0140] Cld.: C, 44.49; H, 6.40; Gd, 20.80; N, 9.26.
[0141] Fnd.: C, 44.21; H, 6.72; Gd, 20.23; N, 9.11.
Example 3
a)
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7-.alpha.,.alp-
ha.',.alpha.''-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,-
10-tetraazacyclododecane
[0142] 19.6 g (50 mmol) of
1-[4-(benzyloxy-carbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyc-
lododecane that is described in Example 1a as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 76.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 500 ml of water each, the organic phase is dried
on magnesium sulfate and evaporated to the dry state. The residue
is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0143] Yield: 41.1 g (76% of theory) of a colorless, crystalline
powder
[0144] Elementary analysis:
[0145] Cld.: C, 72.13; H, 8.10; N, 6.47.
[0146] Fnd.: C, 71.88; H, 8.21; N, 6.25.
b)
10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.-
''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododeca-
ne
[0147] 32.5 g (30 mmol) of the title compound of Example 3a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0148] Yield: 22.0 g (quantitative) of a colorless powder
[0149] Elementary analysis:
[0150] Cld.: C, 61.56; H, 8.80; N, 9.70.
[0151] Fnd.: C, 61.17; H, 8.98; N, 9.41.
c) Gd Complex of the
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-
-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0152] 14.4 g (20 mmol) of the ligand that is described in Example
3b is dissolved in 150 ml of water and 150 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 8 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and evaporated to the dry state.
The residue is taken up with formic acid and evaporated to the dry
state several times with the addition of dichloromethane and then
dried in a vacuum until a constant weight is reached.
[0153] Yield: 12.4 g (65% of theory) of a colorless powder.
[0154] Water content (Karl-Fischer): 8.0%
[0155] Elementary analysis (relative to anhydrous substance):
[0156] Cld.: C, 50.72; H, 6.90; Gd 17.95; N, 7.99.
[0157] Fnd.: C, 51.03; H, 7.08; Gd, 17.42; N, 8.11.
Example 4
a)
10-[4-(t-Butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7-.alpha.,.alph-
a.',.alpha.''-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetr-
aazacyclododecane
[0158] 26.6 g (81.1 mmol) of
N-[2-bromo-2-phenylacetyl]-glycine-t-butylester (Example 6a of WO
98/24775) is added to 27.9 g (162.2 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained
1-[4-(t-butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclo-
dodecane (21.0 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) of
N-ethyldiisopropylamine in 200 ml of dichloromethane are added to
62.45 g (0.2 mol) of 2-(trifluoromethanesulfonyl-oxy)propanoic acid
benzyl ester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3),
360) in 400 ml of dichloromethane, and it is stirred for 6 hours
under reflux and then overnight at room temperature. It is
extracted three times with 500 ml of water each, the organic phase
is dried on magnesium sulfate and evaporated to the dry state. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0159] Yield: 34.0 g (75% of theory) of a colorless, crystalline
powder
[0160] Elementary analysis:
[0161] Cld.: C, 68.93; H, 7.45; N, 7.73.
[0162] Fnd.: C, 69.12; H, 7.57; N, 7.60.
b)
10-(4-(t-Butyloxycarbonyl-1-phenyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alp-
ha.',.alpha.''-trimethyl-1,4,7-tris(carboxy-methyl)-1,4,7,10-tetraazacyclo-
dodecane
[0163] 27.2 g (30 mmol) of the title compound of Example 4a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0164] Yield: 17.5 g (quantitative) of a colorless powder
[0165] Elementary analysis:
[0166] Cld.: C, 55.95; H, 7.13; N, 12.08.
[0167] Fnd.: C, 56.21; H, 6.99; N, 11.83.
c) Gd Complex of
10-(4-carboxy-1-phenyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-
-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0168] 11.6 g (20 mmol) of the t-butylester that is described in
Example 4b is dissolved in a very little trifluoroacetic acid and
stirred for 15 minutes at room temperature. After 250 ml of diethyl
ether is added, it is stirred for 2 more hours, the precipitate is
suctioned off and dried in a vacuum. The thus obtained free ligand
is dissolved in 200 ml of water and 80 ml of isopropanol, set at pH
7 with dilute ammonia and acidified by adding 5 ml of acetic acid.
3.6 g (10 mmol) of gadolinium oxide is added, and it is refluxed
for 3 hours. After complexing is completed, it is set at pH 7.4
again with ammonia and chromatographed on silica gel (mobile
solvent: dichloromethane/methanol/ammonia: 20/20/1). The fractions
that contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0169] Yield: 11.6 g (72% of theory) of a colorless powder.
[0170] Water content (Karl-Fischer): 9.0%
[0171] Elementary analysis (relative to anhydrous substance):
[0172] Cld.: C, 44.19; H, 5.22; Gd 21.43; N, 9.54.
[0173] Fnd.: C, 43.91; H, 5.27; Gd, 21.09; N, 9.77.
Example 5
a) 4-(Ethoxycarbonylmethoxy)-phenylacetic acid methyl ester
[0174] 10 g (60.2 mmol) of hydroxyphenylacetic acid methyl ester
(Aldrich) is dissolved in 75 ml of acetone. 18.4 g (133 mmol) of
solid potassium carbonate is added. 17.8 ml (123 mmol) of
bromoacetic acid ethyl ester is added in drops under reflux within
15 minutes, it is kept at this temperature for another 4 hours, and
it is stirred overnight at room temperature. Precipitate is
filtered out, the solution is evaporated to the dry state and
chromatographed on silica gel (hexane/ethyl acetate 3:1). The
fractions that contain the product are combined and concentrated by
evaporation.
[0175] Yield: 14.6 g (96% of theory)
[0176] Elementary analysis:
[0177] Cld.: C, 61.90; H, 6.39.
[0178] Fnd.: C, 61.67; H, 6.50.
b) .alpha.-Bromo-4-(ethoxycarbonylmethoxy)-phenylacetic acid methyl
ester
[0179] 13.5 g (53.5 mmol) of the title compound of Example 5a is
dissolved in 75 ml of carbon tetrachloride. 9.52 g (53.5 mmol) of
N-bromosuccinimide and 48 mg of dibenzoyl peroxide are added,
refluxed for 5 hours and stirred overnight at room temperature. The
suspension is washed twice with sodium bicarbonate solution and
once with water, the organic phase is dried with magnesium sulfate,
desiccant is suctioned off, and the filtrate is evaporated to the
dry state in a vacuum. The residue is chromatographed on silica gel
(hexane/ethyl acetate 3:1). The fractions that contain the product
are combined and concentrated by evaporation.
[0180] Yield: 15.4 g (87% of theory)
[0181] Elementary analysis:
[0182] Cld.: C, 47.15; H, 4.57; Br, 24.13.
[0183] Fnd.: C, 47.01; H, 4.76; Br, 23.70.
c)
10-[.alpha.-(4-(Ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,-
4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(benzyloxycarbonylmethy-
l)-1,4,7,10-tetraazacyclododecane
[0184] 26.9 g (81.1 mmol) of the bromine compound that is described
in Example 5b above is added to 27.9 g (162.2 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/triethylamine=10/5/0.1). The thus obtained
1-[.alpha.-(4-(ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,4,7-
,10-tetraazacyclododecane (21.1 g; 50 mmol; 62% of theory) and 60
ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml of
dichloromethane are added to 62.45 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)propanoic acid benzyl ester
(Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 ml
of dichloromethane; and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0185] Yield: 34.1 g (75% of theory) of a colorless, crystalline
powder
[0186] Elementary analysis:
[0187] Cld.: C, 67.38; H, 7.10; N, 6.16.
[0188] Fnd.: C, 67.20; H, 7.33; N, 6.31.
d)
10-[.alpha.-(4-(Ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,-
4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,1-
0-tetraazacyclododecane
[0189] 27.3 g (30 mmol) of the title compound of Example 5c is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0190] Yield: 19.3 g (quantitative) of a colorless powder
[0191] Elementary analysis:
[0192] Cld.: C, 56.42; H, 7.26; N, 8.77.
[0193] Fnd.: C, 56.21; H, 7.56; N, 8.47.
e) Gd Complex of
10-[.alpha.-(4-carboxymethoxyphenyl)-carboxymethyl]-1,4,7-.alpha.,.alpha.-
',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane
[0194] 13.3 g (20 mmol) of the title compound of Example 5d is
taken up in 250 ml of 2N sodium hydroxide solution and 250 ml of
tetrahydrofuran, and it is stirred for 5 days at 40.degree. C.
Then, the aqueous phase is set at pH 7 with Amberlite IR-120.RTM.
(H.sup.+ form), 80 ml of isopropanol is added, and it is acidified
by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinum oxide
is added, and it is refluxed for 3 hours. After complexing is
completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0195] Yield: 8.6 g (61% of theory) of a colorless powder.
[0196] Water content (Karl-Fischer): 9.3%
[0197] Elementary analysis (relative to anhydrous substance):
[0198] Cld.: C, 43.19; H, 4.97; Gd, 20.94; N, 7.46.
[0199] Fnd.: C, 43.22; H, 5.29; Gd, 20.42; N, 7.11.
Example 6
a) 4-(Ethoxycarbonylpropoxy)-phenylacetic acid methyl ester
[0200] 10 g (60.2 mmol) of hydroxyphenylacetic acid methyl ester
(Aldrich) is dissolved in 75 ml of acetone. 18.4 g (133 mmol) of
solid potassium carbonate is added. 17.8 ml (123 mmol) of
4-bromobutyric acid ethyl ester is added in drops under reflux
within 15 minutes, and it is kept at this temperature for another 4
hours and stirred overnight at room temperature. Precipitate is
filtered out, the solution is evaporated to the dry state, and it
is chromatographed on silica gel (hexane/ethyl acetate 3:1). The
fractions that contain the product are combined and concentrated by
evaporation.
[0201] Yield: 16.4 g (97% of theory)
[0202] Elementary analysis:
[0203] Cld.: C, 64.27; H, 7.19.
[0204] Fnd.: C, 64.41; H, 6.92.
b) .alpha.-Bromo-[4-(ethoxycarbonylpropoxy)-phenyl]-acetic acid
methyl ester
[0205] 15.0 g (53.5 mmol) of the title compound of Example 6a is
dissolved in 75 ml of carbon tetrachloride. 9.52 g (53.5 mmol) of
N-bromosuccinimide and 48 mg of dibenzoyl peroxide are added, and
it is refluxed for 5 hours and stirred overnight at room
temperature. The suspension is washed twice with sodium bicarbonate
solution and once with water, the organic phase is dried with
magnesium sulfate, desiccant is filtered out, and the filtrate is
evaporated to the dry state in a vacuum. The residue is
chromatographed on silica gel (hexane/ethyl acetate 3:1). The
fractions that contain the product are combined and concentrated by
evaporation.
[0206] Yield: 15.9 g (83% of theory)
[0207] Elementary analysis:
[0208] Cld.: C, 50.16; H, 5.33; Br, 22.24.
[0209] Fnd.: C, 50.33; H, 5.04; Br, 21.94.
c)
10-[.alpha.-(4-(Ethoxycarbonylpropoxy)phenyl)-methoxycarbonylmethyl]-1,-
4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(benzyloxycarbonylmethy-
l)-1,4,7,10-tetraazacyclododecane
[0210] 29.1 g (81.1 mmol) of the bromine compound that is described
in Example 6b above is added to 27.9 g (162.2 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/triethylamine=10/5/0.1). The thus obtained
1-[.alpha.-(4-(ethoxycarbonylpropoxy)phenyl)methoxycarbonyl
methyl]-1,4,7,10-tetraazacyclododecane (22.5 g; 50 mmol; 62% of
theory) and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 62.45 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)propanoic acid-benzyl ester
(Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 ml
of dichloromethane, and it is refluxed for 6 hours and then
overnight at room temperature. It is extracted three times with 500
ml each of water, the organic phase is dried on magnesium sulfate
and evaporated to the dry state. The residue is chromatographed on
silica gel (mobile solvent: dichloromethane/methanol: 20/1). The
fractions that contain the product are combined and concentrated by
evaporation.
[0211] Yield: 30.5 g (65% of theory) of a colorless, crystalline
powder
[0212] Elementary analysis:
[0213] Cld.: C, 67.93; H, 7.31; N, 5.98.
[0214] Fnd.: C, 67.95; H, 7.22; N, 6.13.
d)
10-[.alpha.-(4-(Ethoxycarbonylpropoxy)phenyl)-methoxycarbonylmethyl]-1,-
4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,1-
0-tetraazacyclododecane
[0215] 28.1 g (30 mmol) of the title compound of Example 6c is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0216] Yield: 20.0 g (quantitative) of a colorless powder
[0217] Elementary analysis:
[0218] Cld.: C, 57.64; H, 7.56; N, 8.40.
[0219] Fnd.: C, 57.43; H, 7.77; N, 8.69.
e) Gd Complex of
10-[.alpha.-(4-carboxypropoxyphenyl)-carboxymethyl]-1,4,7-.alpha.,.alpha.-
',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane
[0220] 13.3 g (20 mmol) of the title compound of Example 6d is
taken up in 250 ml of 2N sodium hydroxide solution and 250 ml of
tetrahydrofuran, and it is stirred for 5 days at 40.degree. C.
Then, the aqueous phase is set at pH 7 with Amberlite IR-120.RTM.
(H.sup.+ form), 80 ml of isopropanol is added, and it is acidified
by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide
is added, and it is refluxed for 3 hours. After complexing is
completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0221] Yield: 9.3 g (55% of theory) of a colorless powder.
[0222] Water content (Karl-Fischer): 8.0%
[0223] Elementary analysis (relative to anhydrous substance):
[0224] Cld.: C, 44.72; H, 5.31; Gd, 20.19; N, 7.19.
[0225] Fnd.: C, 44.31; H, 5.88; Gd, 19.93; N, 7.11.
Example 7
a) 4-(Ethoxycarbonyldecyloxy)-phenylacetic acid methyl ester
[0226] 10 g (60.2 mmol) of hydroxyphenylacetic acid methyl ester
(Aldrich) is dissolved in 75 ml of acetone. 18.4 g (133 mmol) of
solid potassium carbonate is added, 36.1 g (123 mmol) of
.omega.-bromoundecanoic acid ethyl ester in 50 ml of acetone is
added in drops, refluxed for 8 hours and stirred overnight at room
temperature. The undissolved material is filtered out, the solution
is evaporated to the dry state and chromatographed on silica gel
(hexane/ethyl acetate 3:1). The fractions that contain the product
are combined and concentrated by evaporation.
[0227] Yield: 20.3 g (89% of theory)
[0228] Elementary analysis:
[0229] Cld.: C, 69.81; H, 9.05.
[0230] Fnd.: C, 69.50; H, 8.91.
b) .alpha.-Bromo-[4-(ethoxycarbonyldecyloxy)-phenyl]-acetic acid
methyl ester
[0231] 20.2 g (53.5 mmol) of the title compound of Example 7a is
dissolved in 75 ml of carbon tetrachloride. 9.52 g (53.5 mmol) of
N-bromosuccinimide and 48 mg of dibenzoyl peroxide are added,
refluxed for 5 hours and stirred overnight at room temperature. The
suspension is washed twice with sodium bicarbonate solution and
once with water, the organic phase is dried with magnesium sulfate,
desiccant is filtered out, and the filtrate is evaporated to the
dry state in a vacuum. The residue is chromatographed on silica gel
(hexane/ethyl acetate 3:1). The fractions that contain the product
are combined and concentrated by evaporation.
[0232] Yield: 21.0 g (86% of theory)
[0233] Elementary analysis:
[0234] Cld.: C, 57.77; H, 7.27; Br, 17.47.
[0235] Fnd.: C, 57.95; H, 7.41; Br, 17.02.
c)
10-[.alpha.-(4-(Ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1-
,4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(benzyloxycarbonylmeth-
yl)-1,4,7,10-tetraazacyclododecane
[0236] 37.1 g (81.1 mmol) of the bromine compound that is described
in Example 7b above is added to 27.9 g (162.2 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/triethylamine=10/5/0.1). The thus obtained
1-[.alpha.-(4-(ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1,4,-
7,10-tetraazacyclododecane (27.4 g; 50 mmol; 62% of theory) and 60
ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml of
dichloromethane are added to 62.45 g (0.2 mmol) of
2-(trifluoromethanesulfonyloxy)propanoic acid benzyl ester
(Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 ml
of dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate, and it is evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0237] Yield: 33.6 g (65% of theory) of a colorless, crystalline
powder
[0238] Elementary analysis:
[0239] Cld.: C, 69.61; H, 7.98; N, 5.41.
[0240] Fnd.: C, 69.75; H, 7.88; N, 5.12.
d)
10-[.alpha.-(4-(Ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1-
,4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,-
10-tetraazacyclododecane
[0241] 31.1 g (30 mmol) of the title compound of Example 7c is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0242] Yield: 23.0 g (quantitative) of a colorless powder
[0243] Elementary analysis:
[0244] Cld.: C, 61.24; H, 8.43; N, 7.32.
[0245] Fnd.: C, 60.96; H, 8.61; N, 7.22.
e) Gd Complex of
10-[.alpha.-(4-carboxydecyloxyphenyl)-carboxymethyl]-1,4,7-.alpha.,.alpha-
.',.alpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodod-
ecane
[0246] 15.3 g (20 mmol) of the title compound of Example 7d is
taken up in 250 ml of 2N sodium hydroxide solution and 250 ml of
tetrahydrofuran, and it is stirred for 5 days at 40.degree. C.
Then, the aqueous phase is set at pH 7 with Amberlite IR-120.RTM.
(H.sup.+ form), 80 ml of isopropanol is added, and it is acidified
by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide
is added, and it is refluxed for 3 hours. After complexing is
completed, it is set at pH 7.4 again with ammonia, and it is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0247] Yield: 11.5 g (60% of theory) of a colorless powder.
[0248] Water content (Karl-Fischer): 8.5%
[0249] Elementary analysis (relative to anhydrous substance):
[0250] Cld.: C, 49.30; H, 6.32; Gd, 17.93; N, 6.39.
[0251] Fnd.: C, 49.56; H, 6.10; Gd, 17.52; N, 6.63.
Example 8
a)
10-(p-Methoxycarbonylbenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-trimethyl-
-1,4,7-tris(benzyloxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane
[0252] 18.6 g (81.1 mmol) of 4-bromomethyl-benzoic acid methyl
ester (Aldrich) in 150 ml of chloroform is added to 27.9 g (162.2
mmol) of 1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of
chloroform, and it is stirred overnight at room temperature. 250 ml
of water is added, the organic phase is separated, and it is washed
twice in each case with 200 ml of water. The organic phase is dried
on magnesium sulfate and evaporated to the dry state in a vacuum.
The residue is chromatographed on silica gel (mobile solvent:
methanol/aqueous 25% ammonia=8/1). The thus obtained
1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane (21.6 g;
67.3 mmol; 83% of theory) and 60 ml (0.35 mol) of
N-ethyldiisopropylamine in 200 ml of dichloromethane are added to
62.45 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid
benzyl ester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3),
360) in 400 ml of dichloromethane, and it is stirred for 6 hours
under reflux and then overnight at room temperature. It is
extracted three times with 500 ml of water each, the organic phase
is dried on magnesium sulfate and evaporated to the dry state. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0253] Yield: 41.8 g (77% of theory) of a colorless, crystalline
powder
[0254] Elementary analysis:
[0255] Cld.: C, 69.95; H, 7.24; N, 6.94.
[0256] Fnd.: C, 69.57; H, 7.39; N, 7.12.
b)
10-(p-Carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-t-
ris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0257] 24.2 g (30 mmol) of the title compound of Example 8a is
dissolved in 400 ml of methanol, mixed with 100 ml of 15N sodium
hydroxide solution, refluxed for 6 hours and stirred overnight at
room temperature. After concentration by evaporation in a vacuum,
the residue is dissolved in 200 ml of water and set at pH 7 by
adding IR-120.RTM. cation exchanger (H.sup.+ form). Exchanger is
filtered out and evaporated to the dry state in a vacuum. The
residue is complexed without being further characterized.
[0258] Thin-layer system: n-butanol/aqueous ammonia/ethanol/water
12/6/3/3
[0259] Yield: 16 g
c) Gd Complex of
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-trimethyl-1,4,7-tri-
s(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0260] 11 g (20 mmol) of the ligand that is described in Example 8b
is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added and refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0261] Yield: 8.9 g (61% of theory) of a colorless powder.
[0262] Water content (Karl-Fischer): 7.2%
[0263] Elementary analysis (relative to anhydrous substance):
[0264] Cld.: C, 44.37; H, 5.21; Gd, 23.23; N, 8.28.
[0265] Fnd.: C, 44.12; H, 5.46; Gd, 22.93; N, 8.51.
Example 9
a)
10-(p-Methoxycarbonylbenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(isop-
ropyl)-1,4,7-tris(benzyloxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane
[0266] 21.6 g (67.3 mmol) of the
1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane that is
described in Example 8a as an intermediate product and 60 ml (0.35
mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane are
added to 85.1 g (0.25 mol) of
2-(trifluoromethanesulfonyloxy)-isovaleric acid benzyl ester
(Walker et al., Tetrahedron (1997), 53(43), 14591) in 400 ml of
dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0267] Yield: 48.5 g (81% of theory) of a colorless, crystalline
powder
[0268] Elementary analysis:
[0269] Cld.: C, 71.43; H, 7.92; N, 6.29.
[0270] Fnd.: C, 71.12; H, 7.79; N, 6.55.
b)
10-(p-Carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(isopropyl)-1-
,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0271] 26.7 g (30 mmol) of the title compound of Example 9a is
dissolved in 400 ml of methanol, mixed with 100 ml of 15N sodium
hydroxide solution, refluxed for 6 hours and stirred overnight at
room temperature. After concentration by evaporation in a vacuum,
the residue is dissolved in 200 ml of water and set at pH 7 by
adding IR-120.RTM. cation exchanger (H.sup.+ form). Exchanger is
filtered out, and it is evaporated to the dry state in a vacuum.
The residue is complexed without being further characterized.
[0272] Thin-layer system: n-butanol/aqueous ammonia/ethanol/water
12/6/3/3
[0273] Yield: 19 g
c) Gd Complex of
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(isopropyl)-1,4-
,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0274] 12.6 g (20 mmol) of the ligand that is described in Example
9b is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia,
and it is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0275] Yield: 10.9 g (65% of theory) of a colorless powder.
[0276] Water content (Karl-Fischer): 9.0%
[0277] Elementary analysis (relative to anhydrous substance):
[0278] Cld.: C, 48.93; H, 6.23; Gd, 20.66; N, 7.36.
[0279] Fnd.: C, 48.87; H, 6.01; Gd, 20.22; N, 7.59.
Example 10
a)
10-(p-Methoxycarbonylbenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(cycl-
ohexyl)-1,4,7-tris(benzyl
oxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane
[0280] 21.6 g (67.3 mmol) of the
1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane that is
described in Example 8a as an intermediate product and 60 ml (0.35
mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane are
added to 95.1 g (0.25 mol) of
2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 500 ml of water each, the organic phase is dried
on magnesium sulfate and evaporated to the dry state. The residue
is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0281] Yield: 48.3 g (71% of theory) of a colorless, crystalline
powder
[0282] Elementary analysis:
[0283] Cld.: C, 73.63; H, 8.17; N, 5.54.
[0284] Fnd.: C, 73.42; H, 8.39; N, 5.75.
b)
10-(p-Carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(cyclohexyl)--
1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0285] 30.3 g (30 mmol) of the title compound of Example 10a is
dissolved in 400 ml of methanol, mixed with 100 ml of 15N sodium
hydroxide solution, refluxed for 6 hours and stirred overnight at
room temperature. After concentration by evaporation in a vacuum,
the residue is dissolved in 200 ml of water and set at pH 7 by
adding IR-120.RTM. cation exchanger (H.sup.+ form). Exchanger is
filtered out, and it is evaporated to the dry state in a vacuum.
The residue is complexed without being further characterized.
[0286] Thin-layer system: n-butanol/aqueous ammonia/ethanol/water
12/6/3/3
[0287] Yield: 22.5 g
c) Gd Complex of
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(cyclohexyl)-1,-
4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0288] 15.0 g (20 mmol) of the ligand that is described in Example
10b is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and evaporated to the dry state.
The residue is taken up with formic acid and evaporated to the dry
state several times with the addition of dichloromethane and then
dried in a vacuum until a constant weight is reached.
[0289] Yield: 11.9 g (63% of theory) of a colorless powder.
[0290] Water content (Karl-Fischer): 7.0%
[0291] Elementary analysis (relative to anhydrous substance):
[0292] Cld.: C, 54.52; H, 6.75; Gd, 17.85; N, 6.36.
[0293] Fnd.: C, 54.19; H, 6.83; Gd, 17.61; N, 6.69.
Example 11
a)
10-(p-Methoxycarbonylbenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-triphenyl-
-1,4,7-tris(benzyloxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane
[0294] 21.6 g (67.3 mmol) of
1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane that is
described in Example 8a as an intermediate product and 60 ml (0.35
mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane are
added to 93.6 g (0.25 mol) of
2-(trifluoromethanesulfonyloxy)-2-phenylacetic acid benzyl ester
(Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 ml
of dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0295] Yield: 50.8 g (76% of theory) of a colorless, crystalline
powder
[0296] Elementary analysis:
[0297] Cld.: C, 74.98; H, 6.49; N, 5.64.
[0298] Fnd.: C, 75.22; H, 6.61; N, 5.47.
b)
10-(p-Carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-triphenyl-1,4,7-t-
ris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0299] 29.8 g (30 mmol) of the title compound of Example 11a is
dissolved in 400 ml of methanol, mixed with 100 ml of 15N sodium
hydroxide solution, refluxed for 6 hours and stirred overnight at
room temperature. After concentration by evaporation in a vacuum,
the residue is dissolved in 200 ml of water and set at pH 7 by
adding IR-120.RTM. cation exchanger (H.sup.+ form). Exchanger is
filtered out, and it is evaporated to the dry state in a vacuum.
The residue is complexed without being further characterized.
[0300] Thin-layer system: n-butanol/aqueous ammonia/ethanol/water
12/6/3/3
[0301] Yield: 22.0 g
c) Gd Complex of
10-(p-carboxybenzyl)-1,4,7-.alpha.,.alpha.',.alpha.''-triphenyl-1,4,7-tri-
s(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0302] 14.6 g (20 mmol) of the ligand that is described in Example
11b is dissolved in 200 ml of water and 80 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and evaporated to the dry state.
The residue is taken up with formic acid and evaporated to the dry
state several times with the addition of dichloromethane and then
dried in a vacuum until a constant weight is reached.
[0303] Yield: 13.1 g (70% of theory) of a colorless powder.
[0304] Water content (Karl-Fischer): 8.1%
[0305] Elementary analysis (relative to anhydrous substance):
[0306] Cld.: C, 55.67; H, 4.79; Gd, 18.22; N, 6.49.
[0307] Fnd.: C, 55.33; H, 4.97; Gd, 17.92; N, 6.54.
Example 12
a)
10-[4-(t-Butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7-.alpha.,.alph-
a.',.alpha.''-triphenyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetr-
aazacyclododecane
[0308] 26.6 g (81.1 mmol) of
N-[2-bromo-2-phenylacetyl]-glycine-t-butylester (Example 6a of WO
98/24775) is added to 27.9 g (162.2 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained
1-[4-(t-butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclo-
dodecane (21.0 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) of
N-ethyldiisopropylamine in 200 ml of dichloromethane are added to
74.9 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)-2-phenylacetic
acid benzyl ester (Qabar et al., Tetrahedron Letters (1998),
39(33), 5895) in 400 ml of dichloromethane, and it is stirred for 6
hours under reflux and then overnight at room temperature. It is
extracted three times with 500 ml of water each, the organic phase
is dried on magnesium sulfate and evaporated to the dry state. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 30/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0309] Yield: 37.7 g (69% of theory) of a colorless, crystalline
powder
[0310] Elementary analysis:
[0311] Cld.: C, 73.67; H, 6.74; N, 6.41.
[0312] Fnd.: C, 73.44; H, 6.43; N, 6.79.
b)
10-(4-(t-Butoxycarbonyl-1-phenyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha-
.',.alpha.''-triphenyl-1,4,7-tris(carboxy-methyl)-1,4,7,10-tetraazacyclodo-
decane
[0313] 32.8 g (30 mmol) of the title compound of Example 12a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0314] Yield: 24.8 g (quantitative) of a colorless powder
[0315] Elementary analysis:
[0316] Cld.: C, 67.22; H, 6.74; N, 8.52.
[0317] Fnd.: C, 67.00; H, 6.85; N, 8.23.
c) Gd Complex of
10-(4-carboxy-1-phenyl-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-
-triphenyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0318] 16.4 g (20 mmol) of the t-butylester that is described in
Example 12b is dissolved in very little trifluoroacetic acid, and
it is stirred for 15 minutes at room temperature. After 250 ml of
diethyl ether is added, it is stirred for 2 more hours, the
precipitate is suctioned off, and it is dried in a vacuum. The thus
obtained free ligand is dissolved in 200 ml of water and 80 ml of
isopropanol, set at pH 7 with dilute ammonia and acidified by
adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide is
added, and it is refluxed for 3 hours. After complexing is
completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 25/15/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0319] Yield: 11.7 g (59% of theory) of a colorless powder.
[0320] Water content (Karl-Fischer): 7.5%
[0321] Elementary analysis (relative to anhydrous substance):
[0322] Cld.: C, 54.83; H, 4.82; Gd, 17.09; N, 7.61.
[0323] Fnd.: C, 54.91; H, 4.67; Gd, 16.62; N, 7.33.
Example 13
a)
10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(isopropyl)-1,4,7-tris(benzyloxy-carbonyl
methyl)-1,4,7,10-tetraazacyclododecane
[0324] 23.2 g (81.1 mmol) of 2-bromoacetylglycine-benzyl ester
(Teger-Nilsson et al., WO 93/11152, page 38) is added to 34.4 g
(0.2 mol) of 1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of
chloroform, and it is stirred overnight at room temperature. 250 ml
of water is added, the organic phase is separated, and it is washed
twice in each case with 200 ml of water. The organic phase is dried
on magnesium sulfate and evaporated to the dry state in a vacuum.
The residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained
1-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecane
(19.6 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) of
N-ethyldiisopropylamine in 200 ml of dichloromethane are added to
68.1 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)-isovaleric acid
benzyl ester (Walker et al., Tetrahedron (1997), 53/43), 14591) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 500 ml of water each, the organic phase is dried
on magnesium sulfate and evaporated to the dry state. The residue
is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0325] Yield: 37.0 g (78% of theory) of a colorless, crystalline
powder
[0326] Elementary analysis:
[0327] Cld.: C, 69.67; H, 7.76; N, 7.39.
[0328] Fnd.: C, 69.51; H, 7.88; N, 7.39.
b)
10-(4-Carboxy-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(i-
sopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0329] 28.4 g (30 mmol) of the title compound of Example 13a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0330] Yield: 17.7 g (quantitative) of a colorless powder
[0331] Elementary analysis:
[0332] Cld.: C, 55.18; H, 8.40; N, 11.92.
[0333] Fnd.: C, 54.97; H, 8.70; N, 11.88.
c) Gd Complex of
10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(iso-
propyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0334] 11.8 g (20 mmol) of the ligand that is described in Example
13b is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0335] Yield: 12.1 g (75% of theory) of a colorless powder.
[0336] Water content (Karl-Fischer): 8.0%
[0337] Elementary analysis (relative to anhydrous substance):
[0338] Cld.: C, 43.71; H, 6.25; Gd, 21.19; N, 9.44.
[0339] Fnd.: C, 43.90; H, 6.40; Gd, 20.80; N, 9.33.
Example 14
a)
10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(cyclohexyl)-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetra-
azacyclododecane
[0340] 18.9 g (50 mmol) of
1-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecane
that is described in Example 13a as an intermediate product and 60
ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml of
dichloromethane are added to 76.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 500 ml of water each, the organic phase is dried
on magnesium sulfate and evaporated to the dry state. The residue
is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0341] Yield: 38.5 g (72% of theory) of a colorless, crystalline
powder
[0342] Elementary analysis:
[0343] Cld.: C, 71.95; H, 8.02; N, 6.56.
[0344] Fnd.: C, 71.90; H, 8.21; N, 6.73.
b)
10-(4-Carboxy-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(c-
yclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0345] 32.1 g (30 mmol) of the title compound of Example 14a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0346] Yield: 21.2 g (quantitative) of a colorless powder
[0347] Elementary analysis:
[0348] Cld.: C, 61.08; H, 8.69; N, 9.89.
[0349] Fnd.: C, 61.27; H, 8.55; N, 9.41.
c) Gd Complex of
10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-.alpha.,.alpha.',.alpha.''-tris(cyc-
lohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0350] 14.2 g (20 mmol) of the ligand that is described in Example
14b is dissolved in 150 ml of water and 150 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 8 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and evaporated to the dry state.
The residue is taken up with formic acid and evaporated to the dry
state several times with the addition of dichloromethane and then
dried in a vacuum until a constant weight is reached.
[0351] Yield: 13.5 g (71% of theory) of a colorless powder.
[0352] Water content (Karl-Fischer): 9.0%
[0353] Elementary analysis (relative to anhydrous substance):
[0354] Cld.: C, 50.15; H, 6.78; Gd, 18.24; N, 8.12.
[0355] Fnd.: C, 49.92; H, 6.51; Gd, 18.01; N, 8.31.
Example 15
a)
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-2,5,8,11-tetrameth-
yl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid-tri-t-butylester, sodium bromide complex
[0356] 0.50 g (1.67 mmol) of 2-bromo-propionylglycine-benzyl ester
(Example 1e of WO 98/24774) is added to 1.14 g (5 mmol) of
2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane (Petrov et al.,
DE 19608307; Ranganathan et al., WO 95/31444), dissolved in 10 ml
of chloroform, and it is stirred overnight at room temperature. 250
ml of water is added, the organic phase is separated, and it is
washed twice in each case with 200 ml of water. The organic phase
is dried on magnesium sulfate and evaporated to the dry state in a
vacuum. The residue is chromatographed on silica gel (mobile
solvent: chloroform/methanol/aqueous 25% ammonia=10/5/1). 822 mg
(4.2 mmol) of bromoacetic acid-tert-butyl ester is added to the
thus obtained
1-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-2,5,8,11-tetramethyl--
1,4,7,10-tetraazacyclododecane (0.70 g; 1.27 mmol; 76% of theory)
and 541 mg (5.1 mmol) of sodium carbonate in 5 ml of acetonitrile,
and it is stirred for 12 hours at 60.degree. C. It is cooled to
0.degree. C., and salts are filtered out. The filtrate is
evaporated to the dry state, and the residue is chromatographed on
silica gel (mobile solvent: methylene chloride/methanol=20:1).
[0357] Yield: 964 mg (85% of theory) of a colorless solid
[0358] Elementary analysis:
[0359] Cld.: C, 56.49; H, 8.01; N, 7.84; Na, 2.57; Br, 8.95.
[0360] Fnd.: C, 56.37; H, 7.88; N, 7.61; Na, 2.33; Br, 8.59.
b)
10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-2,5,8,11-tetramethyl-1,4,7,10--
tetraazacyclododecane-1,4,7-triacetic acid-tri-tert-butyl ester
(sodium bromide complex)
[0361] 893 mg (1.0 mmol) of the title compound of Example 15a is
dissolved in 10 ml of isopropanol, and a spatula tip full of
palladium catalyst (10% Pd/C) is added. It is hydrogenated
overnight at room temperature. Catalyst is filtered out, and the
filtrate is evaporated to the dry state. The residue is
recrystallized from dioxane.
[0362] Yield: 562 mg (70% of theory) of a crystalline solid
[0363] Elementary analysis:
[0364] Cld.: C, 52.36; H, 8.16; N, 8.72; Na, 2.86; Br, 9.95.
[0365] Fnd.: C, 52.51; H, 8.30; N, 8.93; Na, 2.71; Br, 9.44.
c) Gadolinium complex of
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-2,5,8,11-tetramethyl-1,4,7,10-te-
traazacyclododecane-1,4,7-triacetic acid
[0366] 803 mg (1.0 mmol) of the title compound of Example 15b is
dissolved in 5 ml of trifluoroacetic acid and stirred for 3 hours
at room temperature. It is evaporated to the dry state, the residue
is taken up in 300 ml of water, and the solution is added to a
column, filled with Reillex.RTM. 425 PVP. It is eluted with water.
The product-containing fractions are combined and evaporated to the
dry state (446 mg; 0.84 mmol) and again dissolved in 4 ml of water.
152 mg (0.42 mmol) of gadolinium oxide is added, and it is heated
for 3 hours to 90.degree. C. It is evaporated to the dry state
(vacuum), and the residue is crystallized from 90% aqueous ethanol.
The crystals are suctioned off, washed once with ethanol, then with
acetone and finally with dimethyl ether and dried in a vacuum
furnace at 130.degree. C. (24 hours).
[0367] Yield: 469 mg (65% of theory) of a colorless, crystalline
powder
[0368] Water content: 5%
[0369] Elementary analysis (relative to anhydrous substance):
[0370] Cld.: C, 40.28; H, 5.58; N, 10.21; Gd, 22.93.
[0371] Fnd.: C, 40.06; H, 5.75; N, 10.43; Gd, 22.40.
Example 16
Gd Complex of
10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-.alpha.,.alp-
ha.',.alpha.''-tris-(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraaz-
acyclododecane
[0372] 2.27 g (3 mmol) of the Gd complex acid that is described in
Example 2 is dissolved in 15 ml of DMF, mixed with 380 mg (3.3
mmol) of N-hydroxysuccinimide and 681 mg (3.3 mmol) of
dicyclohexylcarbodiimide while being cooled with ice and
preactivated for 1 hour in ice. Then, a mixture that consists of
839 mg (3.3 mmol) of N-(2-aminoethyl)maleimide trifluoroacetate
salt (Arano et al., J. Med. Chem., 1996, 39, 3458) and 0.7 ml (4
mmol) of N,N-diisopropylethyl-amine in 10 ml of DMF is added and
stirred overnight at room temperature. The reaction mixture is
cooled again in an ice bath, filtered, and the filtrate is
evaporated to the dry state in a vacuum. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 1/1).
[0373] Yield: 997 mg (35% of theory)
[0374] Water content (Karl-Fischer): 7.5%
[0375] Elementary analysis (relative to anhydrous substance):
[0376] Cld.: C, 46.51; H, 6.20; Gd, 17.91; N, 11.17.
[0377] Fnd.: C, 46.28; H, 6.44; Gd, 17.31; N, 11.26.
Example 17
Gd Complex of
10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-.alpha.,.alp-
ha.',.alpha.''-tris-(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraa-
zacyclododecane
[0378] 2.63 g (3 mmol) of the Gd complex acid that is described in
Example 3 is dissolved in 15 ml of DMF, mixed with 380 mg (3.3
mmol) of N-hydroxysuccinimide and 681 mg (3.3 mmol) of
dicyclohexylcarbodiimide while being cooled with ice, and
preactivated for 1 hour in ice. Then, a mixture that consists of
839 mg (3.3 mmol) of N-(2-aminoethyl)maleimide trifluoroacetate
salt (Arano et al., J. Med. Chem., 1996, 39, 3458) and 0.7 ml (4
mmol) of N,N-diisopropylethylamine in 10 ml of DMF is added and
stirred overnight at room temperature. The reaction mixture is
cooled again in an ice bath, filtered, and the filtrate is
evaporated to the dry state in a vacuum. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 1/1).
[0379] Yield: 1.24 g (39% of theory)
[0380] Water content (Karl-Fischer): 6.0%
[0381] Elementary analysis (relative to anhydrous substance):
[0382] Cld.: C, 51.74; H, 6.66; Gd, 15.75; N, 9.82.
[0383] Fnd.: C, 51.77; H, 6.41; Gd, 15.25; N, 10.02.
Example 18
a) (3-Bromo-2-oxo-pyrrolidin-1-yl)acetic acid benzyl ester
[0384] 67.7 g (0.2 mol) of glycinebenzyl ester tosylate and 61.2 ml
(0.44 mol) of triethylamine are dissolved in 200 ml of methylene
chloride and added in drops at 0.degree. C. to a solution of 52.9 g
(0.2 mol) of 2,4-dibromobutyric acid chloride (Gramain et al.
Synth. Commun. (1997), (27), 1827) in 200 ml of methylene chloride
within 45 minutes, and it is stirred for 18 hours at room
temperature. The reaction mixture is now added in drops at
0.degree. C. to a solution of 400 ml of aqueous 32% sodium
hydroxide and 2 g of tetrabutylammonium hydrogen carbonate (about
15 minutes), and it is stirred for 30 minutes. Then, the phases are
separated, and the aqueous phase is extracted three times with 200
ml each of dichloromethane. The organic phases are dried on sodium
sulfate, the solution is evaporated to the dry state and
chromatographed on silica gel (methylene chloride). The fractions
that contain the product are combined and concentrated by
evaporation.
[0385] Yield: 29.3 g (47% of theory)
[0386] Elementary analysis:
[0387] Cld.: C, 50.02; H, 4.52; N, 4.49.
[0388] Fnd.: C, 50.34; H, 4.44; N, 4.41.
b)
10-[1-(Benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.a-
lpha.',.alpha.''-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-t-
etraazacyclododecane
[0389] 20.7 (66.3 mmol) of (3-bromo-2-oxo-pyrrolidin-1-yl)acetic
acid benzyl ester is added to 28.7 g (165.8 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained
1-[1-(benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraazacy-
clododecane (20.9 g; 51.8 mmol; 78% of theory) and 60 ml (0.35 mol)
of N-ethyldiisopropylamine in 200 ml of dichloromethane are added
to 62.45 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic
acid benzyl ester (Kitazaki et al., Chem. Pharm. Bull. (1999),
47(3), 360) in 400 ml of dichloromethane, and it is stirred for 6
hours under reflux and then overnight at room temperature. It is
extracted three times with 500 ml of water each, the organic phase
is dried on magnesium sulfate and evaporated to the dry state. The
residue is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0390] Yield: 32.7 g (71% of theory) of a colorless, crystalline
powder
[0391] Elementary analysis:
[0392] Clod.: C, 68.82; H, 7.13; N, 7.87.
[0393] Find.: C, 68.54; H, 7.28; N, 8.01.
c)
10-[1-(Carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0394] 26.7 g (30 mmol) of the title compound of Example 18b is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0395] Yield: 15.8 g (quantitative) of a colorless powder
[0396] Elementary analysis:
[0397] Cld.: C, 52.16; H, 7.42; N, 13.22.
[0398] Fnd.: C, 52.32; H, 7.35; N, 13.11.
d) Gd Complex of
10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.alph-
a.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0399] 10.6 g (20 mmol) of the ligand that is described in Example
18c is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic elate is
freeze-dried.
[0400] Yield: 9.7 g (67% of theory) of a colorless powder.
[0401] Water content (Karl-Fischer): 8.3%
[0402] Elementary analysis (relative to anhydrous substance)
[0403] Cld.: C, 40.40; H, 5.31; Gd, 23.00; N, 10.24.
[0404] Fnd.: C, 39.99; H, 5.55; Gd, 22.93; N, 10.45.
Example 19
a)
10-[1-(Benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.a-
lpha.',.alpha.''-tris(isopropyl)-1,4,7-tris(benzyloxycarbonyl
methyl)-1,4,7,10-tetraazacyclododecane
[0405] 20.2 g (50 mmol) of
1-[1-(benzyloxycarbonylmethyl)-2-oxo-pyrolidin-3-yl]-1,4,7,10-tetraazacyc-
lododecane that is described in Example 18b as an intermediate
product and 60 ml (0.35 mmol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 68.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-isovaleric acid benzyl ester
(Walker et al., Tetrahedron (1997), 53(43), 14591) in 400 ml of
dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0406] Yield: 34.1 g (70% of theory) of a colorless, crystalline
powder
[0407] Elementary analysis:
[0408] Cld.: C, 70.27; H, 7.76; N, 7.19.
[0409] Fnd.: C, 70.45; H, 7.61; N, 7.11.
b)
10-[1-(Carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodod-
ecane
[0410] 29.2 g (30 mmol) of the title compound of Example 19a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0411] Yield: 18.4 g (quantitative) of a colorless powder
[0412] Elementary analysis:
[0413] Cld.: C, 56.75; H, 8.38; N, 11.41.
[0414] Fnd.: C, 56.89; H, 8.31; N, 11.37.
c) Gd Complex of
10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.alph-
a.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
ane
[0415] 12.3 g (20 mmol) of the ligand that is described in Example
19b is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0416] Yield: 11.9 g (75% of theory) of a colorless powder.
[0417] Water content (Karl-Fischer): 8.2%
[0418] Elementary analysis (relative to anhydrous substance):
[0419] Cld.: C, 45.36; H, 6.30; Gd, 20.48; N, 9.12.
[0420] Fnd.: C, 45.89; H, 6.22; Gd, 20.23; N, 9.01.
[0421] The Dy complex of
10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.alph-
a.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
ane is obtained analogously with use of 12.3 g (20 mmol) of the
ligand that is described in Example 19b and 3.73 g (10 mmol) of
dysprosium oxide instead of gadolinium oxide.
[0422] Yield: 11.4 g (71% of theory) of a colorless powder.
[0423] Water content (Karl-Fischer): 8.0%
[0424] Elementary analysis (relative to anhydrous substance):
[0425] Cld.: C, 45.05; H, 6.26; Dy, 21.02; N, 9.06.
[0426] Fnd.: C, 45.35; H, 6.22; Dy, 20.88; N, 9.04.
Example 20
a)
10-[1-(Benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.a-
lpha.',.alpha.''-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,-
7,10-tetraazacyclododecane
[0427] 20.2 g (50 mmol) of
1-[1-(benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraazacy-
clododecane that is described in Example 18b as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 76.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 500 ml of water each, the organic phase is dried
on magnesium sulfate and evaporated to the dry state. The residue
is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0428] Yield: 37.2 g (68% of theory) of a colorless, crystalline
powder
[0429] Elementary analysis:
[0430] Cld.: C, 72.43; H, 8.01; N, 6.40.
[0431] Fnd.: C, 72.55; H, 7.98; N, 6.35.
b)
10-[1-(Carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodo-
decane
[0432] 32.8 g (30 mmol) of the title compound of Example 20a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0433] Yield: 22.0 g (quantitative) of a colorless powder
[0434] Elementary analysis:
[0435] Cld.: C, 62.19; H, 8.65; N, 9.54.
[0436] Fnd.: C, 62.44; H, 8.56; N, 9.46.
c) Gd Complex of
10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.alph-
a.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane
[0437] 14.6 g (20 mmol) of the ligand that is described in Example
20b is dissolved in 150 ml of water and 150 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 8 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and evaporated to the dry state.
The residue is taken up with formic acid and evaporated to the dry
state several times with the addition of dichloromethane, and then
it is dried in a vacuum until a constant weight is reached.
[0438] Yield: 12.1 g (65% of theory) of a colorless powder.
[0439] Water content (Karl-Fischer): 7.0%
[0440] Elementary analysis (relative to anhydrous substance):
[0441] Cld.: C, 51.39; H, 6.81; Gd, 17.70; N, 7.89.
[0442] Fnd.: C, 51.64; H, 6.77; Gd, 17.44; N, 7.77.
Example 21
a) (3-Bromo-2-oxo-pyrrolidin-1-yl)benzoic acid benzyl ester
[0443] 45.5 g (0.2 mol) of 4-aminobeznoic acid benzyl ester and
30.6 ml (0.22 mol) of triethylamine are dissolved in 200 ml of
methylene chloride and added in drops at 0.degree. C. to a solution
of 52.9 g (0.2 mol) of 2,4-dibromobutyric acid chloride (Gramin et
al. Synth. Commun. (1997), (27), 1827) in 200 ml of methylene
chloride within 45 minutes, and it is stirred for 18 hours at room
temperature. The reaction mixture is now added in drops at
0.degree. C. to a solution of 400 ml of aqueous 32% sodium
hydroxide and 2 g of tetrabutylammonium hydrogen carbonate (about
15 minutes), and it is stirred for 30 minutes. Then, the phases are
separated, and the aqueous phase is extracted three times with 200
ml of dichloromethane each. The organic phases are dried on sodium
sulfate, the solution is evaporated to the dry state and
chromatographed on silica gel (methylene chloride). The fractions
that contain the product are combined and concentrated by
evaporation.
[0444] Yield: 38.2 g (51% of theory)
[0445] Elementary analysis:
[0446] Cld.: C, 57.77; H, 4.31; N, 3.74.
[0447] Fnd.: C, 57.99; H, 4.27; N, 3.66.
b)
10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,-
.alpha.',.alpha.''-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-
-tetraazacyclododecane
[0448] 26.9 g (71.9 mmol) of (3-bromo-2-oxo-pyrrolidin-1-yl)benzoic
acid benzyl ester is added to 31.2 g (180 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained
1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraaza-
cyclododecane (26.1 g; 56.1 mmol; 78% of theory) and 60 ml (0.35
mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane are
added to 62.45 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)propanoic acid benzyl ester
(Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 ml
of dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0449] Yield: 36.3 g (68% of theory) of a colorless, crystalline
powder
[0450] Elementary analysis:
[0451] Cld.: C, 70.64; H, 6.88; N, 7.36.
[0452] Fnd.: C, 70.89; H, 6.81; N, 7.29.
c)
10-[1-(4-Carboxyphenyl)-2-oxo-pyrrolidin-3-y]-1,4,7-.alpha.,.alpha.',.a-
lpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0453] 28.6 g (30 mmol) of the title compound of Example 21 b is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0454] Yield: 17.7 g (quantitative) of a colorless powder
[0455] Elementary analysis:
[0456] Cld.: C, 56.84; H, 6.98; N, 11.84.
[0457] Fnd.: C, 57.04; H, 6.91; N, 11.79.
d) Gd Complex of
10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0458] 11.8 g (20 mmol) of the ligand that is described in Example
21c is dissolved in 200 ml of water and 80 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0459] Yield: 11.1 g (71% of theory) of a colorless powder.
[0460] Water content (Karl-Fischer): 7.5%
[0461] Elementary analysis (relative to anhydrous substance):
[0462] Cld.: C, 45.09; H, 5.13; Gd, 21.08; N, 9.39.
[0463] Fnd.: C, 45.45; H, 5.11; Gd, 20.78; N, 9.40.
Example 22
a)
10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,-
.alpha.',.alpha.''-tris(isopropyl)-1,4,7-tris(benzyloxycarbonyl
methyl)-1,4,7,10-tetraazacyclododecane
[0464] 23.3 g (50 mmol) of
1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraaza-
cyclododecane that is described in Example 21b as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 68.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-isovaleric acid benzyl ester
(Walker et al., Tetrahedron (1997), 53(43), 14591) in 400 ml of
dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0465] Yield: 35.3 g (68% of theory) of a colorless, crystalline
powder
[0466] Elementary analysis:
[0467] Cld.: C, 71.86; H, 7.49; N, 6.76.
[0468] Fnd.: C, 71.99; H, 7.46; N, 6.71.
b)
10-[1-(4-Carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.-
alpha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclod-
odecane
[0469] 31.1 g (30 mmol) of the title compound of Example 22a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0470] Yield: 20.2 g (quantitative) of a colorless powder
[0471] Elementary analysis:
[0472] Cld.: C, 60.43; H, 7.90; N, 10.36.
[0473] Fnd.: C, 60.59; H, 7.82; N, 10.31.
c) Gd Complex of
10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodod-
ecane
[0474] 13.5 g (20 mmol) of the ligand that is described in Example
22b is dissolved in 200 ml of water and 80 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0475] Yield: 12.4 g (72% of theory) of a colorless powder.
[0476] Water content (Karl-Fischer): 7.8%
[0477] Elementary analysis (relative to anhydrous substance):
[0478] Cld.: C, 49.20; H, 6.07; Gd, 18.94; N, 8.44.
[0479] Fnd.: C, 49.51; H, 6.04; Gd, 18.71; N, 8.45.
[0480] The Dy complex of
10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodod-
ecane is analogously obtained with use of 13.5 g (20 mmol) of the
ligand that is described in Example 22b and 3.73 g (10 mmol) of
dysprosium oxide instead of gadolinium oxide.
[0481] Yield: 13.0 g (75% of theory) of a colorless powder.
[0482] Water content (Karl-Fischer): 7.5%
[0483] Elementary analysis (relative to anhydrous substance):
[0484] Cld.: C, 48.89; H, 6.03; Dy, 19.45; N, 8.38.
[0485] Fnd.: C, 49.11; H, 6.04; Dy, 19.22; N, 8.36.
Example 23
a)
10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,-
.alpha.',.alpha.''-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,-
4,7,10-tetraazacyclododecane
[0486] 23.3 g (50 mmol) of
1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraaza-
cyclododecane that is described in Example 21b as an intermediate
product and 60 ml (0.35 mol) of N-ethylodiisopropylamine in 200 ml
of dichloromethane are added to 76.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-2-cyclohexyalacetic acid benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 500 ml of water each, the organic phase is dried
on magnesium sulfate and evaporated to the dry state. The residue
is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0487] Yield: 41.1 g (71% of theory) of a colorless, crystalline
powder
[0488] Elementary analysis:
[0489] Cld.: C, 73.74; H, 7.76; N, 6.06.
[0490] Fnd.: C, 73.91; H, 7.69; N, 6.01.
b)
10-[1-(4-Carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.-
alpha.''-tris(cyclohexyl)-1,4,7-tris-(carboxymethyl)-1,4,7,10-tetraazacycl-
ododecane
[0491] 34.7 g (30 mmol) of the title compound of Example 23a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0492] Yield: 23.8 g (quantitative) of a colorless powder
[0493] Elementary analysis:
[0494] Cld.: C, 64.88; H, 8.23; N, 8.80.
[0495] Fnd.: C, 65.04; H, 8.19; N, 8.70.
c) Gd Complex of
10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-.alpha.,.alpha.',.al-
pha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodo-
decane
[0496] 15.9 g (20 mmol) of the ligand that is described in Example
23b is dissolved in 150 ml of water and 150 ml of isopropanol and
acidified by adding 5 ml of acetic acid.
[0497] 3.6 g (10 mmol) of gadolinium oxide is added, and it is
refluxed for 8 hours. After complexing is completed, it is set at
pH 7.4 again with ammonia and chromatographed on silica gel (mobile
solvent: dichloromethane/methanol/ammonia: 20/20/1). The fractions
that contain the product are combined and evaporated to the dry
state. The residue is taken up with formic acid and evaporated to
the dry state several times with the addition of dichloromethane
and then dried in a vacuum until a constant weight is reached.
[0498] Yield: 12.9 g (65% of theory) of a colorless powder.
[0499] Water content (Karl-Fischer): 7.0%
[0500] Elementary analysis (relative to anhydrous substance):
[0501] Cld.: C, 54.35; H, 6.58; Gd, 16.55; N, 7.37.
[0502] Fnd.: C, 54.66; H, 6.57; Gd, 16.32; N, 7.32.
Example 24
a) (3-Bromo-2-oxo-piperidin-1-yl)acetic acid benzyl ester
[0503] 67.7 g (0.2 mol) of glycine benzyl ester tosylate and 61.2
ml (0.44 mol) of triethylamine are dissolved in 200 ml of methylene
chloride and added in drops at 0.degree. C. to a solution of 55.7 g
(0.2 mol) of 2,5-dibromovaleric acid chloride (Okawara et al. Chem.
Pharm. Bull. (1982), (30), 1225) in 200 ml of methylene chloride
within 45 minutes, and it is stirred for 18 hours at room
temperature. The reaction mixture is now added in drops at
0.degree. C. to a solution of 400 ml of aqueous 32% sodium
hydroxide and 2 g of tetrabutylammonium hydrogen carbonate (about
15 minutes), and it is stirred for 30 minutes. Then, the phases are
separated, and the aqueous phase is extracted three times with 200
ml of dichloromethane each. The organic phases are dried on sodium
sulfate, the solution is evaporated to the dry state and
chromatographed on silica gel (methylene chloride). The fractions
that contain the product are combined and concentrated by
evaporation.
[0504] Yield: 33.2 g (51% of theory)
[0505] Elementary analysis:
[0506] Cld.: C, 51.55; H, 4.94; N, 4.29.
[0507] Fnd.: C, 51.86; H, 4.91; N, 4.18
b)
10-[1-(Benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.al-
pha.',.alpha.''-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-te-
traazacyclododecane
[0508] 18.9 g (58 mmol) of (3-bromo-2-oxo-piperidin-1-yl)acetic
acid benzyl ester is added to 30.3 g (175 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained
1-[1-(benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyc-
lododecane (20.3 g; 48.6 mol; 84% of theory) and 60 ml (0.35 mol)
of N-ethyldiisopropylamine in 200 ml of dichloromethane are added
to 62.45 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic
acid benzyl ester (Kitazaki et al., Chem. Pharm. Bull. (1999),
47(3), 360) in 400 ml of dichloromethane, and it is stirred for 6
hours under reflux and then overnight at room temperature. It is
extracted three times with 500 ml of water each, and the organic
phase is dried on magnesium sulfate and evaporated to the dry
state. The residue is chromatographed on silica gel (mobile
solvent: dichloromethane/methanol: 20/1). The fractions that
contain the product are combined and concentrated by
evaporation.
[0509] Yield: 32.5 g (74% of theory) of a colorless, crystalline
powder
[0510] Elementary analysis:
[0511] Cld.: C, 69.08; H, 7.25; N, 7.75.
[0512] Fnd.: C, 69.34; H, 7.19; N, 7.66.
c)
10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0513] 27.1 g (30 mmol) of the title compound of Example 24b is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0514] Yield: 16.3 g (quantitative) of a colorless powder
[0515] Elementary analysis:
[0516] Cld.: C, 53.03; H, 7.60; N, 12.88.
[0517] Fnd.: C, 53.34; H, 7.54; N, 12.79.
d) Gd Complex of
10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]1,4,7-.alpha.,.alpha.',.alpha.-
''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0518] 10.9 g (20 mmol) of the ligand that is described in Example
24c is dissolved in 200 ml of water and 80 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added and refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0519] Yield: 9.6 g (65% of theory) of a colorless powder.
[0520] Water content (Karl-Fischer): 7.2%
[0521] Elementary analysis (relative to anhydrous substance):
[0522] Cld.: C, 41.31; H, 5.49; Gd, 22.53; N, 10.04.
[0523] Fnd.: C, 41.67; H, 5.48; Gd, 22.21; N, 9.97.
Example 25
a)
10-[1-(Benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.al-
pha.',.alpha.''-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,-
10-tetraazacyclododecane
[0524] 20.9 g (50 mmol) of
1-[1-(benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyc-
lododecane that is described in Example 24b as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 68.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-isovaleric acid benzyl ester
(Walker et al., Tetrahedron (1997), 53(43), 14591) in 400 ml of
dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, and the organic phase is dried on
magnesium sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0525] Yield: 36.2 g (73% of theory) of a colorless, crystalline
powder
[0526] Elementary analysis:
[0527] Cld.: C, 70.49; H, 7.85; N, 7.09.
[0528] Fnd.: C, 70.61; H, 7.83; N, 7.01.
b)
10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane
[0529] 29.6 g (30 mmol) of the title compound of Example 25a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0530] Yield: 18.8 g (quantitative) of a colorless powder
[0531] Elementary analysis:
[0532] Cld.: C, 57.40; H, 8.51; N, 11.16.
[0533] Fnd.: C, 57.64; H, 8.45; N, 11.09.
c) Gd Complex of
10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alpha-
.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododeca-
ne
[0534] 12.6 g (20 mmol) of the ligand that is described in Example
25b is dissolved in 200 ml of water and 80 ml of isopropanol and
acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0535] Yield: 11.7 g (71% of theory) of a colorless powder.
[0536] Water content (Karl-Fischer): 8.1%
[0537] Elementary analysis (relative to anhydrous substance):
[0538] Cld.: C, 46.08; H, 6.44; Gd, 20.11; N, 8.96.
[0539] Fnd.: C, 46.34; H, 6.41; Gd, 19.99; N, 8.91.
[0540] The DY complex of
10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alpha-
.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododeca-
ne is analogously obtained with use of 12.6 g (20 mmol) of the
ligand that is described in Example 25b and 3.73 g (10 mmol) of
dysprosium oxide instead of gadolinium oxide.
[0541] Yield: 10.8 g (66% of theory) of a colorless powder.
[0542] Water content (Karl-Fischer): 7.6%
[0543] Elementary analysis (relative to anhydrous substance):
[0544] Cld.: C, 45.77; H, 6.40; Dy, 20.64; N, 8.90.
[0545] Fnd.: C, 46.01; H, 6.46; Dy, 20.34; N, 8.91.
Example 26
a)
10-[1-(Benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.al-
pha.',.alpha.''-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7-
,10-tetraazacyclododecane
[0546] 20.9 g (50 mmol) of
1-[1-(benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyc-
lododecane that is described in Example 24b as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 76.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 500 ml of water each, the organic phase is dried
on magnesium sulfate and evaporated to the dry state. The residue
is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0547] Yield: 39.8 g (72% of theory) of a colorless, crystalline
powder
[0548] Elementary analysis:
[0549] Cld.: C, 72.60; H, 8.09; N, 6.32.
[0550] Fnd.: C, 72.89; H, 7.98; N, 6.27.
b)
10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodod-
ecane
[0551] 33.3 g (30 mmol) of the title compound of Example 26a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0552] Yield: 22.4 g (quantitative) of a colorless powder
[0553] Elementary analysis:
[0554] Cld.: C, 62.63; H, 8.76; N, 9.36.
[0555] Fnd.: C, 62.77; H, 8.71; N, 9.29.
c) Gd Complex of
10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alpha-
.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
ane
[0556] 14.9 g (20 mmol) of the ligand that is described in Example
26b is dissolved in 150 ml of water and 150 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 8 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and evaporated to the dry state.
The residue is taken up with formic acid and evaporated to the dry
state several times with the addition of dichloromethane and then
dried in a vacuum until a constant weight is reached.
[0557] Yield: 12.9 g (68% of theory) of a colorless powder.
[0558] Water content (Karl-Fischer): 7.6%
[0559] Elementary analysis (relative to anhydrous substance):
[0560] Cld.: C, 51.92; H, 6.93; Gd 17.43; N, 7.76.
[0561] Fnd.: C, 52.09; H, 6.88; Gd, 17.21; N, 7.77.
Example 27
a) (3-Bromo-2-oxo-piperidin-1-yl)benzoic acid benzyl ester
[0562] 45.5 g (0.2 mol) of 4-aminobenzoic acid benzyl ester and
30.6 ml (0.22 mol) of triethylamine are dissolved in 200 ml of
methylene chloride and added in drops within 45 minutes at
0.degree. C. to a solution of 55.3 g (0.2 mol) of
2,5-dibromovaleric acid chloride (Okawara et al. Chem. Pharm. Bull.
(1982), (30), 1225) in 200 ml of methylene chloride, and it is
stirred for 18 hours at room temperature. The reaction mixture is
now added in drops at 0.degree. C. to a solution of 400 ml of
aqueous 32% sodium hydroxide and 2 g of tetrabutylammonium hydrogen
carbonate (about 15 minutes), and it is stirred for 30 minutes.
Then, the phases are separated, and the aqueous phase is extracted
three times with 200 ml of dichloromethane each. The organic phases
are dried on sodium sulfate, the solution is evaporated to the dry
state and chromatographed on silica gel (methylene chloride). The
fractions that contain the product are combined and concentrated by
evaporation.
[0563] Yield: 38.8 g (50% of theory)
[0564] Elementary analysis:
[0565] Cld.: C, 58.78; H, 4.67; N, 3.61.
[0566] Fnd.: C, 59.01; H, 4.50; N, 3.59.
b)
10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.-
alpha.',.alpha.''-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10--
tetraazacyclododecane
[0567] 26.6 g (68.5 mmol) of (3-bromo-2-oxo-piperidin-1-yl)benzoic
acid benzyl ester is added to 31.2 g (180 mmol) of
1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform,
and it is stirred overnight at room temperature. 250 ml of water is
added, the organic phase is separated, and it is washed twice in
each case with 200 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqueous 25% ammonia 10/5/1). The thus obtained
1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazac-
yclododecane (27.6 g; 57.5 mmol; 84% of theory) and 60 ml (0.35
mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane are
added to 62.45 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)propanoic acid benzyl ester
(Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 ml
of dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0568] Yield: 39.4 g (71% of theory) of a colorless, crystalline
powder
[0569] Elementary analysis:
[0570] Cld.: C, 70.86; H, 6.99; N, 7.25.
[0571] Fnd.: C 71.11; H, 6.81; N, 7.17.
c)
10-[1-(4-Carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.a-
lpha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0572] 29.0 g (30 mmol) of the title compound of Example 27b is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0573] Yield: 18.1 g (quantitative) of a colorless powder
[0574] Elementary analysis:
[0575] Cld.: C, 57.51; H, 7.16; N, 11.56.
[0576] Fnd.: C, 57.72; H, 7.11; N, 11.50.
d) Gd Complex of
10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
[0577] 12.1 g (20 mmol) of the ligand that is described in Example
27c is dissolved in 200 ml of water and 80 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0578] Yield: 11.4 g (72% of theory) of a colorless powder.
[0579] Water content (Karl-Fischer): 7.1%
[0580] Elementary analysis (relative to anhydrous substance):
[0581] Cld.: C, 45.84; H, 5.31; Gd, 20.69; N, 9.22.
[0582] Fnd.: C, 45.99; H, 5.26; Gd, 20.55; N, 9.21.
Example 28
a)
10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.-
alpha.',.alpha.''-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,-
7,10-tetraazacyclododecane
[0583] 24.0 g (50 mmol) of
1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazac-
yclododecane that is described in Example 27b as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 68.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-isovaleric acid benzyl ester
(Walker et al., Tetrahedron (1997), 53(43), 14591) in 400 ml of
dichloromethane, and it is stirred for 6 hours under reflux and
then overnight at room temperature. It is extracted three times
with 500 ml of water each, the organic phase is dried on magnesium
sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0584] Yield: 37.8 g (72% of theory) of a colorless, crystalline
powder
[0585] Elementary analysis:
[0586] Cld.: C, 72.04; H, 7.58; N, 6.67.
[0587] Fnd.: C, 72.32; H, 7.46; N, 6.59.
b)
10-[1-(4-Carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.a-
lpha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodo-
decane
[0588] 31.5 g (30 mmol) of the title compound of Example 28a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0589] Yield: 20.7 g (quantitative) of a colorless powder
[0590] Elementary analysis:
[0591] Cld.: C, 60.94; H, 8.04; N, 10.15.
[0592] Fnd.: C, 60.87; H, 8.05; N, 10.11.
c) Gd Complex of
10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane
[0593] 13.8 g (20 mmol) of the ligand that is described in Example
28b is dissolved in 200 ml of water and 80 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 3 hours. After
complexing is completed, it is set at pH 7.4 with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an IR-120.RTM.
cation exchange column (H.sup.+ form). The acidic eluate is
freeze-dried.
[0594] Yield: 12.0 g (68% of theory) of a colorless powder.
[0595] Water content (Karl-Fischer): 7.5%
[0596] Elementary analysis (relative to anhydrous substance):
[0597] Cld.: C, 49.80; H, 6.21; Gd, 18.63; N, 8.30.
[0598] Fnd.: C, 49.99; H, 6.17; Gd, 18.51; N, 8.21.
[0599] The Dy complex of
10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane is analogously obtained with use of 13.8 g (20 mmol) of the
ligand that is described in Example 28b and 3.73 g (10 mmol) of
dysprosium oxide instead of gadolinium oxide.
[0600] Yield: 12.4 g (70% of theory) of a colorless powder.
[0601] Water content (Karl-Fischer): 7.5%
[0602] Elementary analysis (relative to anhydrous substance):
[0603] Cld.: C, 49.50; H, 6.17; Dy, 19.13; N, 8.25.
[0604] Fnd.: C, 49.77; H, 6.18; Dy, 18.89; N, 8.27.
Example 29
a)
10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.-
alpha.',.alpha.''-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4-
,7,10-tetraazacyclododecane
[0605] 24.0 g (50 mmol) of
1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazac-
yclododecane that is described in Example 27b as an intermediate
product and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml
of dichloromethane are added to 76.1 g (0.2 mol) of
2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
400 ml of dichloromethane, and it is stirred for 6 hours under
reflux and then overnight at room temperature. It is extracted
three times with 50 ml of water each, the organic phase is dried on
magnesium sulfate and evaporated to the dry state. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0606] Yield: 40.9 g (70% of theory) of a colorless, crystalline
powder
[0607] Elementary analysis:
[0608] Cld.: C, 73.88; H, 7.84; N, 5.98.
[0609] Fnd.: C, 74.12; H, 7.69; N, 5.89.
b)
10-[1-(4-Carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.a-
lpha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclod-
odecane
[0610] 35.1 g (30 mmol) of the title compound of Example 29a is
dissolved in 400 ml of isopropanol, mixed with 40 ml of water, and
3 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state.
[0611] Yield: 24.3 g (quantitative) of a colorless powder
[0612] Elementary analysis:
[0613] Cld.: C, 65.24; H, 8.34; N, 8.65.
[0614] Fnd.: C, 65.48; H, 8.22; N, 8.60.
c) Gd Complex of
10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-.alpha.,.alpha.',.alp-
ha.''-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclodod-
ecane
[0615] 16.2 g (20 mmol) of the ligand that is described in Example
29b is dissolved in 150 ml of water and 150 ml of isopropanol, and
it is acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of
gadolinium oxide is added, and it is refluxed for 8 hours. After
complexing is completed, it is set at pH 7.4 again with ammonia and
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and evaporated to the dry state.
The residue is taken up with formic acid and evaporated to the dry
state several times with the addition of dichloromethane and then
dried in a vacuum until a constant weight is reached.
[0616] Yield: 13.6 g (68% of theory) of a colorless powder.
[0617] Water content (Karl-Fischer): 7.5%
[0618] Elementary analysis (relative to anhydrous substance):
[0619] Cld.: C, 54.81; H, 6.69; Gd, 16.31; N, 7.26.
[0620] Fnd.: C, 55.11; H, 6.57; Gd, 16.09; N, 7.24.
Example 30
a)
1,7-Bis(benzyloxycarbonyl)-4,10-.alpha.,.alpha.'-dimethyl-4,10-bis(t-bu-
toxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane
[0621] 13.8 g (100 mmol) potassium carbonate and 15.3 g (55 mmol)
2-(trifluoromethane-sulfonyloxy)propane acid t-butyl ester (Decicco
et al., Journal of Organic Chemistry 1995, 60, 4782) are added to
11.01 g (25 mmol)
1,7-bis(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (Kovacs
et al., Synthesis 1997, 759), dissolved in 150 ml of
tetrahydrofurane and 15 ml of water, and it is stirred over night
at room temperature. After evaporation to the dry state, the
residue is distributed between 100 ml dichloromethane and 100 ml of
water, the organic phase is separated and it is washed twice in
each case with 50 ml of water. The organic phase is dried on
magnesium sulfate and evaporated to the dry state in a vacuum. The
residue is chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqu. 25% ammonia=10/5/1). The fractions that
contain the product are combined and concentrated by
evaporation.
[0622] Yield: 17.4 g (86% of theory) of a colorless powder
[0623] Elementary analysis:
[0624] Cld.: C, 65.49; H, 8.10; N, 8.04.
[0625] Fnd.: C, 65.22; H, 8.17; N, 8.20.
b)
1,7-.alpha.,.alpha.'-Dimethyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,10--
tetraazacyclododecane
[0626] 13.9 g (20 mmol) of the title compound of example 30a is
dissolved in 250 ml of isopropanol, mixed with 25 ml of water, and
2 g of palladium catalyst (10% Pd/C) is added. It is hydrogenated
for 8 hours at 50.degree. C. Catalyst is filtered out, and the
filtrate is evaporated to the dry state in a vacuum.
[0627] Yield: 8.5 g (quantitative) of a colorless powder
[0628] Elementary analysis:
[0629] Cld.: C, 61.65; H, 10.35; N, 13.07.
[0630] Fnd.: C, 61.17; H, 10.55; N, 12.85.
c)
1,7-.alpha.,.alpha.'-Dimethyl-1,7-bis-(t-butoxycarbonylmethyl)-4-.alpha-
.-isopropyl-4-benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane
[0631] 8.51 g (25 mmol) 2-(trifluoromethane-sulfonyloxy)-isovaleric
acid benzyl ester (Walker et al., Tetrahedron (1997), 53(43),
14591) in 60 ml of dichloromethane is added to 8.5 g (20 mmol) of
that in example 30b described
1,7-.alpha.,.alpha.'-dimethyl-1,7-bis-(t-butoxycarbonylmethyl)--
1,4,7,10-tetraazacyclododecane and 6.86 ml (40 mmol)
N-ethyldiisopropylamine in 200 ml of dichloromethane and it is
stirred for 6 hours under reflux and then over night at room
temperature. It is extracted three times in each case with 50 ml of
water, the organic phase is dried on magnesium sulfate and
evaporated to the dry state. The residue is chromatographed on
silica gel (mobile solvent: dichloromethane/methanol: 20/1). The
fractions that contain the product are combined and concentrated by
evaporation.
[0632] Yield: 8.8 g (71% of theory) of a colorless powder
[0633] Elementary analysis:
[0634] Cld.: C, 65.99; H, 9.45; N, 9.05.
[0635] Fnd.: C, 66.08; H, 9.25; N, 8.85.
d)
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-.alpha.,.alpha-
.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-isopropyl-4-benzylox-
ycarbonylmethyl)-1,4,7,10-tetraazacyclododecane
[0636] 2.5 g (8.11 mmol) 2-bromopropionylglycin-benzyl ester
(example 1e of WO 98/24774) is added to 4.33 g (7 mmol) of that in
example 30c described
1,7-.alpha.,.alpha.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-
-.alpha.-isopropyl-4-benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododeca-
ne, dissolved in 50 ml of chloroform, and it is stirred at
50.degree. C. over night. 50 ml of water are added, the organic
phase is separated and it is washed twice in each case with 20 ml
of water. The organic phase is dried on magnesium sulfate and
evaporated to the dry state in a vacuum. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0637] Yield: 3.1 g (53% of theory) of a colorless powder
[0638] Elementary analysis:
[0639] Cld.: C, 65.92; H, 8.54; N, 8.36.
[0640] Fnd.: C, 65.77; H, 8.62; N, 8.20.
e) Gd-complex of
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,7-.alpha.,.alpha.'-dimethyl-1,-
7-bis-(carboxymethyl)-4-.alpha.-isopropyl-4-carboxymethyl)-1,4,7,10-tetraa-
zacyclododecane
[0641] 2.5 g (3 mmol) of the title compound of example 30d is
dissolved in 100 ml of isopropanol, mixed with 5 ml of water and
after addition of a spatula tip of palladium catalyst (10% Pd/C),
it is hydrogenated for 8 hours at room temperature. Catalyst is
filtered out, and the filtrate is evaporated to the dry state in a
vacuum. The residue is dissolved in 25 ml of trifluoroacetic acid
without further purification and it is stirred for 2 hours at room
temperature. The deprotected product is precipitated by the
addition of diethylether, exhausted and dried in a vacuum. The
residue is dissolved in 20 ml of water and 2 ml of isopropanol and
the pH is adjusted to 5. 543 mg (1.5 mmol) gadolinium oxide is
added and it is refluxed for 3 hours. After the complexation is
finished, the pH is adjusted to 7.4 with ammonia and it is
chromatographed on silica gel (mobile solvent: dichloromethane
methanol/ammonia: 20/20/1). The fractions that contain the product
are combined and added via an IR-120.RTM.-cation exchange column
(H.sup.+-form). The acidic eluate is freeze-dried.
[0642] Yield: 1.44 g (62% of theory) of a colorless powder. Water
content (Karl-Fischer): 9.3%
[0643] Elementary analysis (referenced to the anhydrous
substance):
[0644] Cld.: C, 41.19; H, 5.76; Gd, 22.47; N, 10.01.
[0645] Fnd.: C, 40.88; H, 5.88; Gd, 22.11; N, 9.79.
The dysprosium complex is obtained analog to this by using 559 mg
(1.5 mmol) dysprosium oxide instead of gadolinium oxide:
[0646] Yield: 1.44 g (62% of theory) of a colorless powder. Water
content (Karl-Fischer): 9.0%
[0647] Elementary analysis (referenced to the anhydrous
substance):
[0648] Cld.: C, 40.88; H, 5.72; Dy 23.05; N, 9.93
[0649] Fnd.: C, 40.58; H, 5.90; Dy, 22.73; N, 9.86.
Example 31
a)
1,7-Bis(benzyloxycarbonyl)-4,10-.alpha.,.alpha.'-diisopropyl-4,10-bis(t-
-butoxycarbonyl methyl)-1,4,7,10-tetraazacyclododecane
[0650] 13.8 g (100 mmol) potassium carbonate and 16.85 g (55 mmol)
2-(trifluoromethane-sulfonyloxy)-isovaleric acid t-butyl ester
(Semmelhack et al., Tetrahedron Letters 34, 1395 (1993)) are added
to 11.01 g (25 mmol)
1,7-bis(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (Kovacs
et al., Synthesis 1997, 759), dissolved in 150 ml of
tetrahydrofurane and 15 ml of water and it is stirred over night at
room temperature. It is evaporated to the dry state in a vacuum and
the residue is distributed between 100 ml of dichloromethane and
100 ml of water, the organic phase is separated and washed twice
with each 50 ml of water. The organic phase is dried on magnesium
sulfate and evaporated to the dry state in a vacuum. The residue is
chromatographed on silica gel (mobile solvent:
chloroform/methanol/aqu. 25% ammonia=10/5/1). The fractions that
contain the product are combined and concentrated by
evaporation.
[0651] Yield: 13.9 g (74% of theory) of a colorless powder.
[0652] Elementary analysis:
[0653] Cld.: C, 66.99; H, 8.57; N, 7.44.
[0654] Fnd.: C, 66.58; H, 8.72; N, 7.22.
b)
1,7-.alpha.,.alpha.'-Diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,-
10-tetraazacyclododecane
[0655] 15.06 g (20 mmol) of the title compound of example 31a is
dissolved in 250 ml of isopropanol, 25 ml of water is added and 2 g
palladium catalyst (10% Pd/C) are added. It is hydrogenated for 8
hours at 50.degree. C. Catalyst is filtered out, and the filtrate
is evaporated to the dry state in a vacuum.
[0656] Yield: 9.7 g (quantitative) of a colorless powder.
[0657] Elementary analysis:
[0658] Cld.: C, 64.43; H, 10.81; N, 11.56.
[0659] Fnd.: C, 64.30; H, 10.94; N, 11.31.
c)
1,7-.alpha.,.alpha.'-Diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alp-
ha.-methyl-4-benzyloxycarbonyl-methyl-1,4,7,10-tetraazacyclododecane
[0660] 8.18 g (25 mmol) 2-(trifluoromethane-sulfonyloxy)propanoic
acid-benzyl ester (Kitazaki et al., Chem. Pharm. Bull. (1999),
47(3), 360) in 60 ml of dichloromethane is added to 8.5 g (20 mmol)
of that in example 31b described
1,7-.alpha.,.alpha.'-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,10-
-tetraazacyclododecane and 6.86 ml (40 mmol)
N-ethyldiisopropylamine in 200 ml of dichloromethane, and it is
stirred for 6 hours under reflux and then over night at room
temperature. It is extracted three times in each case with 50 ml of
water, the organic phase is dried on magnesium sulfate and
evaporated to the dry state. The residue is chromatographed on
silica gel (mobile solvent: dichloromethane/methanol: 20/1). The
fractions that contain the product are combined and concentrated by
evaporation.
[0661] Yield: 9.8 g (76% of theory) of a colorless powder.
[0662] Elementary analysis:
[0663] Cld.: C, 66.84; H, 9.66; N, 8.66.
[0664] Fnd.: C, 66.58; H, 9.82; N, 8.63.
d)
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-.alpha.,.alpha-
.'-diisopropyl-1,7-bis(t-butoxycarbonyl
methyl)-4-.alpha.-methyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclo-
dodecane
[0665] 2.5 g (8.11 mmol) 2-bromopropionylglycin-benzyl ester
(example 1e of WO 98/24774) is added to 4.53 g (7 mmol) of that in
example 31c described
1,7-.alpha.,.alpha.'-diisopropyl-1,7-bis(t-butoxycarbonylmethyl-
)-4-.alpha.-methyl-4-benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododeca-
ne, dissolved in 50 ml of chloroform, and it is stirred at
50.degree. C. over night. 50 ml of water is added, the organic
phase is separated and washed twice in each case with 20 ml of
water. The organic phase is dried on magnesium sulfate and
evaporated to the dry state in a vacuum. The residue is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol: 20/1). The fractions that contain the
product are combined and concentrated by evaporation.
[0666] Yield: 3.6 g (59% of theory) of a colorless powder.
[0667] Elementary analysis:
[0668] Cld.: C, 66.56; H, 8.73; N, 8.09.
[0669] Fnd.: C, 66.26; H, 8.98; N, 7.91.
e) Gd-complex of
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,7-.alpha.,.alpha.'-diisopropyl-
-1,7-bis(carboxymethyl)-4-.alpha.-methyl-4-carboxymethyl-1,4,7,10-tetraaza-
cyclododecane
[0670] 2.6 g (3 mmol) of the title compound of example 31d is
dissolved in 100 ml of isopropanol, 5 ml of water is added and
after addition of a spatula tip of palladium catalyst (10% Pd/C),
it is hydrogenated for 8 hours at room temperature. Catalyst is
filtered out, and the filtrate is evaporated to the dry state in a
vacuum. The residue is dissolved in 25 ml of trifluoroacetic acid
without further purification and it is stirred for 2 hours at room
temperature. The deprotected product is precipitated by addition of
diethylether, exhausted and dried in a vacuum. The residue is
dissolved in 20 ml of water and 2 ml of isopropanol and the pH is
adjusted to 5.543 mg (1.5 mmol) gadolinium oxide is added and it is
refluxed for 3 hours. After the complexation is finished, the pH is
adjusted to 7.4 with ammonia and it is chromatographed on silica
gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).
The fractions that contain the product are combined and added via
an IR-120.RTM.-cation exchange column (H.sup.+-form). The acidic
eluate is freeze-dried.
[0671] Yield: 1.55 g (64% of theory) of a colorless powder.
[0672] Water content (Karl-Fischer): 10.0%
[0673] Elementary analysis (referenced to the anhydrous
substance):
[0674] Cld.: C, 42.90; H, 6.09; Gd, 21.60; N, 9.62.
[0675] Fnd.: C, 42.75; H, 5.93; Gd, 21.21; N, 9.54.
Example 32
a)
1,7-.alpha.,.alpha.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-
-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane
[0676] 9.51 g (25 mmol)
2-(trifluoromethane-sulfonyloxy)-2-cyclohexyl acetic acid-benzyl
ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in
60 ml of dichloromethane is added to 8.5 g (20 mmol) of that in
example 30b described
1,7-.alpha.,.alpha.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,10-te-
traazacyclododecane and 6.86 ml (40 mmol) of
N-ethyldiisopropylamine in 200 ml dichloromethane and it is stirred
for 6 hours under reflux and then over night at room temperature.
It is extracted three times with in each case 50 ml of water, the
organic phase is dried on magnesium sulfate and evaporated to the
dry state. The residue is chromatographed on silica gel (mobile
solvent: dichloromethane/methanol: 20/1). The fractions that
contain the product are combined and concentrated by
evaporation.
[0677] Yield: 9.1 g (69% of theory) of a colorless powder.
[0678] Elementary analysis (referenced to the anhydrous
substance):
[0679] Cld.: C, 67.44; H, 9.48; N, 8.50.
[0680] Fnd.: C, 67.28; H, 9.66; N, 8.31.
b)
10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-.alpha.,.alpha-
.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-cyclohexyl-4-benzylo-
xycarbonylmethyl-1,4,7,10-tetraazacyclododecane
[0681] 2.5 g (8.11 mmol) 2-bromopropionylglycin-benzyl ester
(example 1e of WO 98/24774) is added to 4.61 g (7 mmol) of that in
example 32a described
1,7-.alpha.,.alpha.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-
-.alpha.-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododeca-
ne, dissolved in 50 ml of chloroform, and it is stirred over night
at 50.degree. C. 50 ml of water is added, the organic phase is
separated and it is washed twice in each case with 20 ml of water.
The organic phase is dried on magnesium sulfate and evaporated to
the dry state in a vacuum. The residue is chromatographed on silica
gel (mobile solvent: dichloromethane/methanol: 20/1). The fractions
that contain the product are combined and concentrated by
evaporation.
[0682] Yield: 3.5 g (57% of theory) of a colorless powder
[0683] Elementary analysis:
[0684] Cld.: C, 67.02; H, 8.61; N, 7.97.
[0685] Fnd.: C, 66.86; H, 8.62; N, 8.11.
c) Gd complex of the
10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,7-.alpha.,.alpha.'-dimethyl-1,-
7-bis(carboxymethyl)-4-.alpha.-cyclohexyl-4-carboxymethyl-1,4,7,10-tetraaz-
acyclododecane
[0686] 2.6 g (3 mmol) of the title compound of example 32b is
dissolved in 100 ml of isopropanol, 5 ml of water is added and
after addition of a spatula tip of palladium catalyst (10% Pd/C),
it is hydrogenated for 8 hours at room temperature. Catalyst is
filtered off and the filtrate is evaporated to the dry state in a
vacuum. The residue is dissolved in 25 ml of trifluoroacetic acid
without further purification and it is stirred for two hours at
room temperature. The deprotected product is precipitated by
addition of diethylether, exhausted and dried in a vacuum. The
residue is dissolved in 20 ml of water and 2 ml of isopropanol and
the pH is adjusted to 5. 543 mg (1.5 mmol) gadolinium oxide are
added and it is refluxed for 3 hours. After the complexation is
finished, the pH is adjusted to 7.4 with ammonia and it is
chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an
IR-120.RTM.-cation exchange column (H.sup.+-form). The acidic
eluate is freeze-dried.
[0687] Yield: 1.56 g (64% of theory) of a colorless powder
[0688] Water content (Karl-Fischer): 9.1%
[0689] Elementary analysis (referenced to the anhydrous
substance):
[0690] Cld.: C, 43.83; H, 5.99; Gd, 21.25; N, 9.46.
[0691] Fnd.: C, 43.62; H, 6.18; Gd, 21.04; N, 9.31.
Example 33
a) 10-[.alpha.-(4-(Ethoxycarbonylmethoxy)phenyl)-methoxycarbonyl
methyl]-1,7-.alpha.,.alpha.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.-
alpha.-isopropyl-4-benzyloxycarbonyl
methyl-1,4,7,10-tetraazacyclododecane
[0692] 2.69 g (8.11 mmol) of that in example 5b described
.alpha.-bromo-4-(ethoxycarbonylmethoxy)-phenylacetic acid methyl
ester is added to 4.33 g (7 mmol) of that in example 30c described
1,7-.alpha.,.alpha.'-dimethyl-1,7-bis-(t-butoxycarbonylmethyl)-4-.alpha.--
isopropyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,
dissolved in 50 ml of chloroform, and it is stirred over night at
50.degree. C. 50 ml of water is added, the organic phase is
separated and it is washed twice in each case with 20 ml of water.
The organic phase is dried on magnesium sulfate and evaporated to
the dry state in a vacuum. The residue is chromatographed on silica
gel (mobile solvent: dichloromethane/methanol: 20/1). The fractions
that contain the product are combined and concentrated by
evaporation.
[0693] Yield: 3.5 g (58% of theory) of a colorless powder
[0694] Elementary analysis:
[0695] Cld.: C, 64.95; H, 8.35; N, 6.45.
[0696] Fnd.: C, 64.73; H, 8.22; N, 6.58.
b) Gd-complex of the
10-[.alpha.-(4-carboxymethoxyphenyl)-carboxymethyl]-1,7-.alpha.,.alpha.'--
dimethyl-1,7-bis(carboxymethyl)-4-.alpha.-isopropyl-4-carboxymethyl-1,4,7,-
10-tetraazacyclododecane
[0697] 2.6 g (3 mmol) of the title compound of example 33a is
dissolved in 100 ml of isopropanol, 5 ml of water is added and
after addition of a spatula tip of palladium catalyst (10% Pd/C),
it is hydrogenated for 8 hours at 50.degree. C. Catalyst is
filtered off, the filtrate is evaporated to the dry state in a
vacuum. The residue is treated with 40 ml of a 2N sodium hydroxide
solution and 40 ml tetrahydrofuran, without further purification
and it is stirred for 5 days at 40.degree. C. Afterwards the pH is
adjusted to 7 with Amberlite IR-120.RTM. (H.sup.+-form) and
acidified by addition of 1 ml acetic acid. Then 543 mg (1.5 mmol)
gadolinium oxide is added and it is refluxed for 3 hours. After the
complexation is finished the pH is adjusted to 7.4 with ammonia and
it is chromatographed on silica gel (mobile solvent:
dichloromethane/methanol/ammonia: 20/20/1). The fractions that
contain the product are combined and added via an
IR-120.RTM.-cation exchange column (H.sup.+-form). The acidic
eluate is freeze-dried.
[0698] Yield: 1.82 g (71% of theory) of a colorless powder
[0699] Water content (Karl-Fischer): 8.9%
[0700] Elementary analysis (referenced to the anhydrous
substance):
[0701] Cld.: C, 44.72; H, 5.31; Gd, 20.19; N, 7.19.
[0702] Fnd.: C, 44.88; H, 5.21; Gd, 20.01; N, 6.95.
Example 34
a)
10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-.alpha.-.alpha.'-diisop-
ropyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-methyl-4-benzyloxycarbonyl-
methyl-1,4,7,10-tetraazacyclododecane
[0703] 2.32 g (8.11 mmol) 2-bromoacetylglycin-benzyl ester
(Teger-Nilsson et al., WO 93/11152, page 38) is added to 4.53 g (7
mmol) of that in example 31c described
1,7-.alpha.-.alpha.'-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha-
.-methyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,
dissolved in 50 ml of chloroform, and it is stirred over night at
50.degree. C. 50 ml of water is added, the organic phase is
separated and washed twice in each case with 20 ml of water. The
organic phase is dried on magnesium sulfate and evaporated to the
dry state in a vacuum. The residue is chromatographed on a silica
gel (mobile solvent: dichloromethane/methanol: 20/1). The fractions
that contain the product are combined and concentrated by
evaporation.
[0704] Yield: 4.8 g (81% of theory) of a colorless powder.
[0705] Elementary analysis:
[0706] Cld.: C, 66.25; H, 8.64; N, 8.22.
[0707] Fnd.: C, 66.00; H, 8.56; N, 8.31.
b) Gd-complex of the
10-(4-carboxy-2-oxo-3-azabutyl)-1,7-.alpha.,.alpha.'-diisopropyl-1,7-bis(-
carboxymethyl)-4-.alpha.-methyl-4-carboxymethyl-1,4,7,10-tetraazacyclodode-
cane
[0708] 2.55 g (3 mmol) of the title compound of example 34a is
dissolved in 100 ml of isopropanol, 5 ml of water is added and
after addition of a spatula tip palladium catalyst (10% Pd/C) it is
hydrogenated for 8 hours a room temperature. Catalyst is filtered
off, and the filtrate is evaporated to the dry state in a vacuum.
The residue is dissolved in 25 ml of trifluoroacetic acid without
further purification and it is stirred for 2 hours at room
temperature. The deprotected product is precipitated by addition of
diethylether, exhausted and dried in a vacuum. The residue is
dissolved in 20 ml of water and 2 ml of isopropanol and the pH is
adjusted to 5. 543 mg (1.5 mmol) gadolinium oxide is added and it
is refluxed for 3 hours. After the complexation is finished the pH
is adjusted to 7.4 with ammonia and it is chromatographed on silica
gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).
Fractions that contain the product are combined and added via an
IR-120.RTM.-cation exchange column (H.sup.+-form). The acidic
eluate is freeze-dried.
[0709] Yield: 1.51 g (64% of theory) of a colorless powder.
[0710] Water content (Karl-Fischer): 9.0%
[0711] Elementary analysis (referenced to the anhydrous
substance):
[0712] Cld.: C, 42.06; H, 5.93; Gd, 22.03; N, 9.81.
[0713] Fnd.: C, 41.95; H, 5.99; Gd, 21.77; N, 9.62.
Example 35
a)
10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-.alpha.-.alpha.'-dimeth-
yl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-cyclohexyl-4-benzyloxycarbony-
lmethyl-1,4,7,10-tetraazacyclododecane
[0714] 2.32 g (8.11 mmol) 2-bromoacetylglycin-benzyl ester
(Teger-Nilsson et al., WO 93/11152, page 38) is added to 4.61 g (7
mmol) of that in example 32a described
1,7-.alpha.,.alpha.'-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-.alpha.-c-
yclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,
dissolved in 50 ml of chloroform, and it is stirred over night at
50.degree. C. 50 ml of water is added, the organic phase is
separated and washed twice in each case with 20 ml of water. The
organic phase is dried on magnesium sulfate and evaporated to the
dry state in a vacuum. The residue is chromatographed on silica gel
(mobile solvent: dichloromethane/methanol 20/1). The fractions that
contain the product are combined and concentrated by
evaporation.
[0715] Yield: 4.35 g (72% of theory) of a colorless powder.
[0716] Elementary analysis:
[0717] Cld.: C, 66.72; H, 8.51; N, 8.10.
[0718] Fnd.: C, 66.43; H, 8.77; N, 8.02.
b) Gd-complex of the
10-(4-carboxy-2-oxo-3-azabutyl)-1,7-.alpha.,.alpha.'-dimethyl-1,7-bis(car-
boxymethyl)-4-.alpha.-cyclohexyl-4-carboxymethyl-1,4,7,10-tetraazacyclodod-
ecane
[0719] 2.6 g (3 mmol) of the title compound of example 35a is
dissolved in 100 ml of isopropanol, 5 ml of water is added and
after the addition of a spatula tip of palladium catalyst (10%
Pd/C) it is hydrogenated for 8 hours at room temperature. Catalyst
is filtered out, the filtrate is evaporated to the dry state in a
vacuum. The residue is dissolved in 25 ml of trifluoroacetic acid
without further purification and it is stirred for 2 hours at room
temperature. The deprotected product is precipitated by the
addition of diethylether, exhausted and dried in a vacuum. The
residue is dissolved in 20 ml of water and 2 ml of isopropanol and
the pH is adjusted to 5.543 mg (1.5 mmol) gadolinium oxide is added
and it is refluxed for 3 hours. After the complexation is finished,
the pH is adjusted to 7.4 with ammonia and it is chromatographed on
silica gel (mobile solvent: dichloromethane/methanol/ammonia:
20/20/1). The fractions that contain the product are combined and
added via an IR-120.RTM.-cation exchange column (H.sup.+-form). The
acidic eluate is freeze-dried.
[0720] Yield: 1.74 g (73% of theory) of a colorless powder.
[0721] Water content (Karl-Fischer): 8.8%
[0722] Elementary analysis (referenced to the anhydrous
substance):
[0723] Cld.: C, 43.02; H, 5.83; Gd, 21.66; N, 9.65.
[0724] Fnd.: C, 42.87; H, 6.05; Gd, 21.29; N, 9.55.
Examples 36-108
[0725] Examples 36-108 describe conjugates of the above-described
gadolinium complexes with biomolecules. The conjugates were
produced according to the following general operating instructions
I-IV. The results are summarized in Table 1. Here, "AAV" stands for
general operating instructions, "ACTH" stands for
adrenocorticotropic hormone, and "RP-18" refers to a "reversed
phase" stationary chromatography phase. The number of complexes per
biomolecule was determined by means of ICP (inductively coupled
plasma atomic emission spectroscopy).
General Operating Instructions (AAV) I: Albumin-Amide
Conjugates
[0726] 3 mmol of the Gd complex acid is dissolved in 15 ml of DMF,
mixed with 380 mg (3.3 mmol) of N-hydroxysuccinimide and 681 mg of
dicyclohexylcarbodiimide while being cooled with ice, and
preactivated for 1 hour in ice. The active ester mixture is added
in drops within 30 minutes in a solution of 16.75 g (0.25 mmol) of
bovine serum albumin (BSA) or human serum albumin (HSA) in 150 ml
of phosphate buffer (pH 7.4) and stirred for 2 hours at room
temperature. The batch solution is filtered, the filtrate is
ultrafiltered with an AMICON.RTM. YM30 (cut-off 30,000 Da), the
retentate is chromatographed on a Sephadex.RTM. G50-column, and the
product fractions are freeze-dried.
General Operating Instructions (AAV) II: Albumin-Maleimide
Conjugates
[0727] 0.0438 mmol of the Gd-complex maleimide in 1 ml of DMF is
added to 0.84 g (0.0125 mmol) of bovine serum albumin (BSA),
dissolved in 15 ml of phosphate buffer (pH 7.4), and it is stirred
for one hour at room temperature. The batch solution is filtered,
the filtrate is ultrafiltered with an AMICON.RTM. YM30 (cut-off
30,000 Da), the retentate is chromatographed on a Sephadex.RTM. G50
column, and the product fractions are freeze-dried.
General Operating Instructions (AAV) III: Production of Amide
Conjugates
[0728] 3 mmol of the Gd-complex acid is dissolved in 15 ml of DMF,
mixed with 380 mg (3.3 mmol) of N-hydroxysuccinimide and 681 mg of
dicyclohexylcarbodiimide while being cooled with ice, and
preactivated for 1 hour in ice. The active ester mixture is added
in drops to a solution of 2.5 mmol of amine components in 15-150 ml
of DMF and stirred overnight at room temperature. The batch
solution is filtered and chromatographed on silica gel.
General Operating Instructions (AAV) IV: Production of Maleimido-SH
Conjugates
[0729] 3 mmol of the Gd-complex maleimide in 15 ml of DMF is added
in drops to 2.5 mmol of SH components in 15-150 ml of DMF, and it
is stirred for one hour at room temperature. The batch solution is
chromatographed on silica gel. TABLE-US-00001 TABLE 1 Edukt Gd-
Anzahl Komplexe Komplex pro Biomolekul Ausbeute Beispiel (Beispiel
Nr.) konjugiert mit (Herkunft) AAV (ICP) Bemerkungen (%) 36 1 BSA
Sigma I 3.7 -- quant. 37 2 BSA Sigma I 6.1 -- quant. 38 3 BSA Sigma
I 2.9 -- quant. 39 4 BSA Sigma I 3.5 -- quant. 40 5 BSA Sigma I 4.2
-- quant. 41 6 BSA Sigma I 6.5 -- quant. 42 7 BSA Sigma I 5.0 --
quant. 43 16 BSA Sigma II 0.71 -- quant. 44 17 BSA Sigma II 0.55 --
quant. 45 8 BSA Sigma I 3.0 -- quant. 46 9 BSA Sigma I 4.7 --
quant. 47 10 BSA Sigma I 5.1 -- quant. 48 11 BSA Sigma I 2.7 --
quant. 49 12 BSA Sigma I 4.0 -- quant. 50 13 BSA Sigma I 3.3 --
quant. 51 14 BSA Sigma I 5.8 -- quant. 52 15 BSA Sigma I 4.6 --
quant. 53 18 BSA Sigma I 3.7 -- quant. 54 19 BSA Sigma I 4.1 --
quant. 55 20 BSA Sigma I 2.8 -- quant. 56 21 BSA Sigma I 3.5 --
quant. 57 22 BSA Sigma I 3.3 -- quant. 58 23 BSA Sigma I 2.9 -- 59
24 BSA Sigma I 4.0 -- quant. 60 25 BSA Sigma I 3.5 -- quant. 61 26
BSA Sigma I 3.0 -- quant. 62 27 BSA Sigma I 3.9 -- quant. 63 28 BSA
Sigma I 3.1 -- quant. 64 29 BSA Sigma I 3.4 -- quant. 65 11
(D-Lys16)-ACTH (1-24 human) BACHEM I 2.0 -- quant. 66 12 ACTH
(1-17) BACHEM I 1.7 -- quant. 67 14 H-.beta.-Ala-Phe BACHEM III 1.0
wurde an RP-18 95 gereinigt 68 8 Anti-Inflamatory Peptide 2 BACHEM
I 1.0 -- quant. 69 9 L-Carnosin BACHEM III 1.0 wurde an RP-18 97
gereinigt 70 16 Homoglutathion BACHEM IV 1.0 wurde an RP-18 94
gereinigt 71 17 Guanyl-Cys-OH BACHEM IV 1.0 wurde an RP-18 93
gereinigt 72 8 H-DL-d-Hydroxy-DL-Lys-OH BACHEM III 1.0 wurde an
RP-18 85 gereinigt 73 7 H-.beta.-Ala-Lys-OH BACHEM III 1.0 wurde an
RP-18 87 gereinigt 74 16 H-Arg-Gly-Asp-Cys-OH BACHEM III 1.0 wurde
an RP-18 91 gereinigt 75 9 H-Asp-Leu-Trp-Gln-Lys-OH BACHEM III 1.0
wurde an RP-18 94 gereinigt 76 12 H-Ala-His-Lys-OH BACHEM III 2.0
wurde an RP-18 91 gereinigt 77 13 Endothelin-2 (Human) BACHEM I
0.87 -- quant. 78 14 Human Serumalbumin BACHEM I 5.1 -- quant. 79 7
Human Serumalbumin BACHEM I 3.1 -- quant. 80 8 Human Serumalbumin
BACHEM I 2.3 -- quant. 81 17 Thioguanosin Aldrich IV 1.0 wurde an
RP-18 96 gereinigt 82 5 6-Aminopenicilinsaure Aldrich III 1.0 wurde
an RP-18 92 gereinigt 83 11 4-Aminopteroylglutaminsaure Aldrich III
1.0 wurde an RP-18 65 gereinigt 84 4 2-Amino-purinthiol Aldrich IV
1.0 wurde an RP-18 94 gereinigt 85 12 5-Azacytidin Aldrich III 1.0
wurde an RP-18 96 gereinigt 86 17 4,5-Diamino-2,6- Aldrich IV 1.0
wurde an RP-18 71 dimercaptopyrimidin gereinigt 87 13 Mitomycin C
Aldrich III 1.0 wurde an RP-18 81 gereinigt 88 12 Muraminsaure
Aldrich III 1.0 wurde an RP-18 92 gereinigt 89 6 Puromycin SIGMA
III 1.0 wurde an RP-18 90 gereinigt 90 11 Doxorubicin SIGMA III 1.0
wurde an RP-18 89 gereinigt 91 12 Spectinomycin SIGMA III 1.0 wurde
an RP-18 88 gereinigt 92 4 Streptomycin SIGMA III 1.0 wurde an
RP-18 62 gereinigt 93 14 Neomycin B SIGMA III 1.0 wurde an RP-18 52
gereinigt 94 8 Nystatin SIGMA III 1.0 wurde an RP-18 72 gereinigt
95 3 Hygromycin SIGMA III 1.0 wurde an RP-18 71 gereinigt 96 2
Ampicillin SIGMA III 1.0 wurde an RP-18 42 gereinigt 97 30 BSA
Sigma I 2.1 -- quant. 98 31 BSA Sigma I 1.8 -- quant 99 32 BSA
Sigma I 2.7 -- quant 100 33 BSA Sigma I 1.2 -- quant 101 34 BSA
Sigma I 1.6 -- quant 102 35 BSA Sigma I 2.5 -- quant 103 30 HSA
Sigma I 1.9 -- quant 104 31 HSA Sigma I 2.5 -- quant 105 32 HSA
Sigma I 2.2 -- quant 106 33 HSA Sigma I 1.5 -- Quant 107 34 HSA
Sigma I 1.7 -- quant 108 35 HSA Sigma I 2.4 -- quant #[Key to Table
1:] Beispiel = Example Edukt Gd-Komplex (Beispiel Nr.) = Gd-Complex
Educt (Example No.) konjugiert mit = Conjugated with (Herkunft) =
(Origin) Anzahl Komplexe pro Biomolekul = Number of complexes per
biomolecule Bemerkungen = Remarks Ausbeute (%) = Yield (%)
L-Carnosin = L-Carnosine Homoglutathion = Homoglutathione wurde an
RP-18 gereinigt = was purified on RP-18 Thioguanosin =
Thioguanosine 6-Aminopenicilinsaure = 6-Aminopenicillic acid
4-Aminopteroylglutaminsaure = 4-Aminopteroylglutamic acid
2-Amino-purinthiol = 2-amino-purinethiol 5-Azacytidin =
5-Azacytidine 4,5-Diamino-2,6-dimercaptopyrimidin =
4,5-Diamino-2,6-dimercaptopyridimidine Muraminsaure = Muramic
acid
Example 109
[0730] In this example, the relaxivities of the conjugates from
Examples 36-38 and 97-108 were compared with the relaxivities of
two comparison substances. As comparison substances, Gd-DTPA (1)
with the formula: ##STR16## and Gd-GlyMeDOTA (2) with the formula:
##STR17## which were reacted in each case with bovine serum albumin
(BSA), were used.
[0731] The measurements were made in each case in aqueous solution
and in plasma at +37.degree. C. and a frequency of 20 MHz. The
results are summarized in Table 2 below, whereby the indicated
relaxivities per mol of gadolinium were calculated from the
measured values: TABLE-US-00002 TABLE 2 Gd-Komplex Anzahl Gd/BSA
R.sub.1 (H.sub.2O) R.sub.1 (Plasma) Beispiel (aus Beispiel) or HSA
(L/mmol * s) (L/mmol * s) 36 1 3.7 22.1 25.3 37 2 6.1 29.8 35.7 38
3 2.9 38.2 51.5 39 4 3.5 27.1 29.7 40 5 4.2 20.0 22.4 41 6 6.5 23.2
25.8 42 7 5.0 31.1 37.4 43 16 0.71 38.0 38.3 44 17 0.55 40.6 41.4
97 30 2.1 35.3 39.1 98 31 1.8 32.2 36.5 99 32 2.7 36.5 40.1 100 33
1.2 40.0 44.6 101 34 1.6 31.8 34.8 102 35 2.5 40.1 45.7 103 30 1.9
34.6 39.9 104 31 2.5 31.7 36.1 105 32 2.2 35.4 40.3 106 33 1.5 41.6
46.7 107 34 1.7 33.6 37.0 108 35 2.4 39.1 44.9 Vergleichssubstanz 1
Gd-DTPA 36 13.39 13.97 Vergleichssubstanz 2 Gd-GlyMeDOTA 4.7 18.3
20.8 [Key:] Beispiel = Example Gd-Komplex (aus Beispiel) = Gd
complex (from Example) Anzahl Gd/BSA = Gd/BSA number
Vergleichssubstanz = Comparison substance
[0732] This example shows that the conjugates that are produced
with the compounds according to the invention have, surprisingly
enough, a higher relaxivity than the comparison substances despite
their low number of gadolinium atoms per biomolecule. Compared to
comparison substance 2, it was possible to increase the relaxivity
by the special liganding of the macrocyclic ring.
[0733] The entire disclosures of all applications, patents and
publications, cited herein and of corresponding German application
No. 10135356.1, filed Jul. 20, 2001 is incorporated by reference
herein.
[0734] The preceding examples can be repeated with similar success
by substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
[0735] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention
and, without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
* * * * *
References