NO174394B - Polymer complex consisting of at least one ion of an element of the order numbers 21-29, 42, 44 or 57-83 and a carboxylic acid group containing complexing polymer, preparation thereof, use thereof and diagnostic agent - Google Patents

Abstract

Polymers consisting of a ligand containing carboxylic acid groups and optionally of at least one ion of an element of atomic numbers 21- 29, 42, 44 or 57-83 and optionally cations of inorganic and/or organic bases, amino acids or amino acid amides are valuable complexing agents and complexes for diagnostics.

Description

The present invention relates to a polymer complex consisting of at least one ion of an element of the order numbers 21-

29, 42, 44 or 57-83 and a carboxylic acid group-containing complex forming polymer, as well as methods for preparing such complexes.

The use of complexing agents or complexes or their salts in medicine has long been known. As examples to be mentioned: Complex formers known as stabilizers of pharmaceutical preparations, complexes and their salts as aids for the supply of poorly soluble ions (e.g. iron), complex formers and complexes (preferably calcium or zinc), possibly as salts with inorganic and/or organic bases, as an antidote for detoxification by inadvertent ingestion of heavy metals or their radioactive isotopes and complex formers as aids in nuclear medicine using radioactive isotopes

9 9m

as Tc for the sémtigraphx.

In patent document DE-OS 3401052, paramagnetic complex salts have recently been proposed as diagnostics, mainly as NMR diagnostics.

These complexes or complex salts are quite well tolerated and provide a largely complete excretion of paramagnetic ions. The downside, however, is that they just spread out

non-specifically in the extracellular space and therefore only in exceptional cases sufficient for recognition of pathologically changed tissue.

The attempt to solve at least one part of these problems

by using complex formers, which on the one hand are bound by ionic binding to the possibly suitable metal (see below) as well as on the other hand by binding to a functional group or a non-toxic and most possible organ-specific molecule as a carrier molecule , has so far only had very limited success.

Thus, for example, the number is paramagnetic

centers in the complexes, which are described in the European patent applications No. 88 695 and 150 884, not sufficient for organ-specific imaging.

If the number of necessary metal ions is increased by several times the introduction of complex-forming units into a macromolecule, this is always associated with an intolerable influence on the affinity and/or specificity of this macromolecule [J. Nucl. With. 24, 1158 (1983)].

There is therefore a need for many kinds of purposes for stable, well-soluble, but also more tolerable, easily accessible complex compounds, which contain the largest possible number of necessary metal ions in the complex, without their affinity and/or specificity being lost. It is thus a task for the invention to provide these complex compounds, as well as to provide the simplest possible method for their production. This task is solved with the help of the invention.

It was found that polymer complexes, consisting of a ligand containing a carboxylic acid group, and equipped with amide, hydrazide and/or alkylated or acylated imino subunits, at least one ion of an element with the order numbers 21 - 29, 42 , 44 or 57 - 83 as well as possibly cations of inorganic and/or organic bases, amino acids or amino acid amides, surprisingly, are excellently suited for the preparation of NMR, X-ray and ultrasound diagnostics, as above all in the complex they contain stably bound the necessary number of metal ions for this application.

The polymer complexes according to the invention are characterized by the fact that the complex-forming structure has the general formula I

where A and A' each mean

the meaning of the digits 0 or 1,

s means the whole numbers from 7 to 20,000,

t means the whole numbers from 0 to 20,000,

U means a direct bond, the group

V, S 1 S 2 , S 3 and S 4 each mean a direct bond or a linear, branched, saturated or unsaturated C 1 -C 10 alkylene group which optionally contains an imino, phenylene, phenyleneoxy, phenyleneimino, amide, hydrazide, ester group (r), oxygen, sulfur and/or nitrogen atom(s) and optionally substituted with hydroxy, mercapto, imino, epoxy, oxo, thioxo and/or amino group(s),

K means a complexing agent of the general formulas IA, IB

or IC:

whereby

n and m each stand for the digits 0, 1, 2, 3 or 4, whereby n and

m together will not be more than 4,

k stands for the digits 1, 2, 3, 4 or 5,

1 stands for the digits 0, 1, 2, 3, 4 or 5,

q stands for the digits 0, 1 or 2,

X independently each stands for the residues - C00H or V where

V' means the residue V which exhibits at the end a functional group or a bio- or macromolecule which is bound via this functional group, whereby, if the molecule contains V, at least 0.1% of the substituent

X stands for V,

B, D and E, as the same or different, each stand for the group

with

R2 in the sense of hydrogen or a linear, branched, saturated or unsaturated C^-C^ alkyl group which optionally contains oxygen and/or nitrogen atom(s) and is optionally substituted with hydroxy and/or amino group(s) ,

u in the sense of the digits 0, 1, 2, 3, 4 or 5,

v in the sense of the digits 0 or 1,

whereby B, D and E each contain a minimum of 2 and a maximum of 5 carbon atoms,

Z stands for the group

or the residue X, R<1> stands for the direct bond or a hydrogen atom, with the proviso that Z only stands for the group

when R<1> simultaneously means a hydrogen atom, and that Z only stands for the residue X, when R<1> simultaneously means a direct

bond,

W means a hydrogen atom, biotin, avidin, avidin-biotin-antibody, avidin-biotin-antibody fragments, the group Uw-Vw-K„, wherein Uw, Vw and K„ each have one of the meanings mentioned for U, V and K,

V or the group

whereby free COOH groups which are not required for complex formation of the metal ions of the elements with the specified ordinal numbers, if desired are present as a salt of an inorganic and/or organic base, an amino acid or an amino acid amide, or as an ester and/or amide.

Preferred polymer units are the complex-forming structures with the general formula Ia:

If the polymer complex according to the invention is to be used in NMR diagnostics, the central ion in the complex salt must be paramagnetic. These are especially the divalent and trivalent ions of the elements with ordinal numbers 21-29, 42, 44 and 57-70. Suitable ions are, for example, chromium(III), manganese(II), iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III ), the samarium(III) and ytterbium(III) ion. Due to their very strong magnetic moment, the gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III) and haem(III) ions are particularly preferred.

If the polymer complex according to the invention is to be used in X-ray diagnostics, the central ion must be derived from an element with a higher order number, in order to achieve sufficient absorption of the X-rays. It was found that for this purpose diagnostic agents, which contain a physiologically compatible complex salt with central ions of elements with ordinal numbers between 21-29, 42, 44, 52-83, are suitable. These are, for example, the lanthanum(III) ion and the above-mentioned ions of the lanthanum family.

The polymer complexes according to the invention contain at least one ion of an element with the above-mentioned order number.

The alkylene group that stands for V as well as the alkyl group that stands for R 2 , R and R' can be linear, branched, cyclic, aliphatic, aromatic or arylaliphatic and have up to 20 carbon atoms. Preferred are linear mono- to decamethylene groups as well as C-^-C^-alkylenephenyl groups.

For clarification, the following alkylene groups must be mentioned as examples:

Preferred functional groups located at the end of the V"-alkylene group are, for example, maleimidbbenzoyl-, 3-sulfomaleimidobenzoyl-, 4-<maleimidomethyl)-cyclohexylcarbonyl-, 4- [3-sulfo-(maleimidomethyl)-cyclohexyl-carbonyl-, 4 -(p-male-imidophenyl)-butyryl-, 3-(2-pyridyldithio)propionyl-, metha-• cryloyl-(pentamethylene)amido-, bromoacetyl-, iodoacetyl-, 3-iodo-propyl-, 2-bromomethyl- , 3-mercaptopropyl-, 2-mercaptoethyl-, phenylene isothiocyanate-, 3-aminopropyl-, benzyl ester-, ethyl ester-, t-butyl ester-, amino-, C^-Cg-alkylamino-, aminocarbonyl-, hydrazino-, hydrazinocarbonyl-, maleimido-, methacrylamido-, metha-•cryloylhydrazinocarbonyl-, maleimimidamidocarbonyl-, halogen-, mercapto-, hydrazinotrimethylenehydrazinocarbonyl-, aminodimethylene-amidocarbonyl-, bromocarbonyl-, phenylenediazonium-, isothiocyanate-, semicarbazide-, thiosemicarbazide group.

For clarification, some selected groups are listed:

where R and R' are the same or different and each represents a hydrogen atom, a saturated or unsaturated C 1 -C 4 -alkyl radical which is optionally substituted with a phenyl group, or a phenyl group.

As examples of the complexing residue K, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, trans-1,2-cyclohexanediaminetetraacetic acid, 1,4,7,10-tetraazacyclododecanetetraacetic acid, 1,4,7-triaza-cyclononane-triacetic acid, 1,4, The 8,11-tetraazatetradecanetetraacetic acid and the 1,5,9-triaza-cyclododecanetriacetic acid are mentioned, which are bonded via a carbon atom or a carbonyl group (each contained in K) to the residues in the polymer units. If desired, part of the carboxylic acids can be present as residues and/or amide.

As polymers which are suitable for the production of the polymer complexes according to the invention, mention should be made, for example, of polyethyleneimine, polylysine, polyaspartic acid, polyethyleneimine-polyacetic acid ester and polyacrylic ester.

The remaining acidic hydrogen atoms, i.e. those that have not been substituted with the central ion, can possibly be completely or partially replaced by cations of inorganic and/or organic bases or amino acids. The corresponding acid groups can also be completely or partially transferred to esters or amides.

Suitable inorganic cations are, for example, the lithium ion, the potassium ion, the calcium ion and especially the sodium ion. Suitable cations of organic bases are among others those of primary, secondary or tertiary amines, such as for example ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethylglucamine and especially N-methylglucamine. Suitable cations of amino acids are, for example, the cations from lysine, arginine and ornithine as well as the amines of otherwise acidic or neutral amino acids.

Suitable esters are preferably those with a C 1 -C 8 alkyl residue. Mention should be made, for example, of the methyl, ethyl and tertiary butyl residue.

If the carboxylic acid groups are to be at least partially present as amines, tertiary amines are preferred. As leftovers come on

these are saturated, unsaturated, linear or branched or cyclic hydrocarbons with up to 5 C atoms, which are optionally substituted with 1 to 3 hydroxy or C 1 -C 4 -alkyl groups. Examples include methyl-, ethyl-, 2-hydroxyethyl-, 2-hydroxy-l-(hydroxymethyl)-ethyl-, 1-(hydroxymethyl)-ethyl-, propyl-, iso-propenyl-, 2-hydroxypropyl-, 3 -hydroxypropyl-, 2,3-dihydroxy-propyl-, butyl-, isobutyl-, isobutenyl-, 2-hydroxybutyl-, 3-hydroxybutyl-, 4-hydroxybutyl-, 2-, 3- and 4-hydroxy-2-methyl -

the butyl, 2- and 3-hydroxyisobutyl, 2,3,4-trihydroxybutyl, 1,2,4-trihydroxybutyl, pentyl, cyclopentyl and 2-methoxyethyl group. The amide residue can also be a heterocyclic 5- or 6-ring which is formed during inclusion of the amine nitrogen. Examples should be mentioned: the pyrrolidinyl, piperidyl, pyrazolidinyl, pyrrolinyl, pyrazolinyl, piperazinyl, morpholinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl ring.

The polymer complexes according to the invention contain

the large number of metal ions necessary for their use, stably bound in the complex.

Thus, for example, equilibrium or re-complexation investigations with the gadolinium complex of the diethylenetriaminepentaacetic acid DTPA (EP 71 564) which can be considered the state of the art and which is recognized in the field as a good contrast agent, that the polymer complexes according to the invention are surprisingly more stable than this over the entire molecular range ( 5 - 2000 kD) which is determined by the index numbers.

The compatibility of the polymer complex is also an example

superior compared to organ-specific monomeric complexes.

The value of the magnitude of the relaxivity, which is a measure of image formation, is surprisingly high: It is a factor 3 - 1000 higher for the polymer complexes according to the invention than for gadolinium-DTPA. In order to achieve a specific signal strength during image formation, a correspondingly smaller molar amount of complex is therefore necessary compared to the monomers.

As a further important advantage of the polymer complexes according to the invention, their excretion ratio must be stated. The secretion rate that is desired depending on the purpose of use can be set very purposefully and easily by means of the molecular weight that is selected.

The polymer complexes according to the invention exhibit a surprisingly high tissue specificity. Thus, for example, already a few minutes after intravenous injection of an N-methyl-glucamine salt solution of the gadolinium(III) complex of polyethyleneimine-poly-DTPA (see example 1), a clear contrast enhancement is achieved in the peripheral tumor tissue in the nuclear resonance image, which persists for a long time. and brings a clear diagnostic benefit.

Surprisingly, with the help of the polymer complexes according to the invention, blood vessels can also be detected without the use of special pulse sequences in vivo, so that they can, among other things, find use as perfusion agents. The production of the polymers according to the invention takes place by polymers which are

equipped with amino-, imino- and/or

, where Fl stands for a leaving group, by alkylation, acylation, amidation and/or hydrazineation are converted into compounds, which as polymer units exhibit structures with the general formula I'

where

A, A<1>, U, V, S^, S2, S^, S4, s and t have the above-mentioned meaning and K<1> are complex formers with the general formulas I'A, I'B, I' C and I'D which are identical to the formulas given for IA, IB, IC and ID, but which instead of the substituents X and Z each have X' and Z<1>, whereby

X' independently of each other stand for -COOH and

Z' stands for the group

or the residue X', with the proviso that part of the COOH groups are present as ester and/or amide if desired. W means a hydrogen atom, the group Uw-Vw-K'w, <h>wherein Uw and V"w have the meaning mentioned above and K' has the meaning mentioned for K, V" or the group

, whereby V" stands for a residue that is shown

at the end of a functional group,

these are then, in a manner known per se, if desired, reacted with at least one metal oxide or metal salt of an element with the ordinal numbers 21 - 29, 42, 44 or 57 - 83 and, if desired, K<1> and/or W' are transferred by conversion of at least one of the -C02H groups contained in K<1> or W to the desired alkylene group which exhibits a functional group at the end and optionally then connection with a macro or biomolecule and/or by binding to a biotin or avidin residue is transferred to K and W, whereby the stated reac-

ion step (with the exception of the macro- or biomolecule attachment, which can only take place after the generation of the functional group) can be carried out in any order, and acidic hydrogen atoms still present in the thus obtained polymer complexes can then optionally be substituted with cations of inorganic and/or or organic bases, amino acids or amino acid amides, respectively the corresponding acid groups can be completely or partially transferred to esters or amides.

Thereby starting with polymers such as polyethyleneimine, polyethyleneimine polyacetic acid derivatives, polyacryl

esters or polylysine, which optionally contain

where Fl stands for a leaving group such as Cl, Br, J, NH2, OCH3, OC2H5, OCH2C6H5, OC3H7, mesylate or tosylate.

These compounds are amidated, hydrazined (Houben-Weyl, Methoden der"organischen Chemie, vol. VIII/3 Georg Thieme Verlag, Stuttgart (1952), 654 and 676), acylated (J. March, Advanced Organic Chemistry, McGraw-Hill, 2nd ed. ., (1977) 377 - 382) and/or alkylated (Houben-Weyl, Methoden der organischen Chemie, volume VI/3 Georg Thieme Verlag, Stuttgart (1965), 187) in a manner known per se.

As a substrate for the introduction of the complex-forming units K-V or K -V serves compounds of the general formulas I'A, I'B, I'C, I' 'AB and,I"C where V'' stands for a substituent which can be converted to V or V'' and Z' stands for an activated carbonyl group.

Examples of an activated carbonyl group include anhydride, p-nitrophenyl ester and acid chloride.

The alkylation or acylation that is carried out to introduce the complexing units is carried out with reagents,

which contains the desired K-V- or K -V -substituents (bonded

w w

to a leaving group) or from which the desired substituent, possibly after modification by subsequent reactions, is generated by the reaction. Halogenides, mesylates, tosylates and anhydrides should be mentioned as examples of the former. The second group includes, for example, oxiranes, thiiranes, aziranes, α,|3-unsaturated carbonyl compounds or their vinylogs, aldehydes, ketones, isothiocyanates and isocyanates.

As examples of subsequent reactions, mention must be made of ester cleavages, hydrogenations, esterifications, oxidations, etherifications and alkylations, which are carried out according to literature methods known to the person skilled in the art.

The compounds I<1> which are required as educts are known (e.g. EP patent application publication number 0154788) or can be prepared from the corresponding polyamines (whereby functional groups present are optionally protected) by alkylation with an ester of the general formula II

where Hal stands for chlorine, bromine or iodine and Y for an acid protecting group.

The reaction takes place in polar, aprotic solvents such as dimethylformamide, dimethylsulfoxide or hexamethylphosphoric acid triamide in the presence of an acid scavenger, such as e.g. tertiary amine (e.g. triethylamine, trimethylamine, N,N-dimethyl-aminopyridine, 1,5-diazabicyclo[4.3.0]-nonen-5(DBN), 1,5-diaza-bicyclo[5.4.0]- undecene-5-(DBU), alkali, alkaline earth carbonate or hydrogen carbonate (e.g. sodium, magnesium, calcium, barium, potassium carbonate and hydrogen carbonate) at temperatures between -10° C and 120° C, preferably between 0 and 50° C.

Acid protecting groups Y include lower alkyl, aryl and aralkyl groups, for example methyl, ethyl, propyl, butyl, phenyl, benzyl, diphenylmethyl, triphenylmethyl, bis-(p-nitrophenyl)methyl, as well as trialkylsilyl groups.

The removal of the protective groups Y takes place using methods known to those skilled in the art, for example by hydrolysis, alkaline saponification of the ester in alkali in an aqueous-alcoholic solution at temperatures from 0 to 50° C or in the case of tert-butyl esters using trifluoroacetic acid.

The preparation of the activated carbonyl derivatives 1<1>'

(e.g. mixed anhydride, N-hydroxysuccinimide esters, acylimidazoles, trimethylsilyl esters) takes place according to methods known from the literature [Houben-Weyl, Methoden der organischen Chemie, Georg Thieme Verlag, Stuttgart, volume E 5(1985), 633 ; Org. React. 12, 157 (1962) ] or described in the experimental part.

The corresponding polyamines which are

necessary for the preparation of the polyamine polyacids of the general formula I'A as the educt, are prepared analogously with methods known from the literature (e.g. Canadian patent no. 1 178 951, Eur. I, Med. Chem. -CMm . Ther. 1985,20,509 and 1986, 21,333), starting from amino acids, which are transferred to optionally ethyleneamine-substituted amides (e.g. with N-(2-aminoethyl)-carbamic acid benzyl ester) and then reduced (possibly after removal of the protective groups) to the desired amines (preferably with diborane or lithium aluminum hydride).

If the polyamine educts for the compounds of the general formula I'B are to be synthesized, it is necessary before the reduction to substitute such an amide by reaction with, for example, ethyloxamate in a polar solvent such as tetrahydrofuran, dimethylsulfoxide or dimethoxyethane at a temperature between 50 and 250° C, preferably 70 to 150° C (possibly in a pressure vessel) to the α-amino group, so that a 3-aza-2-oxo-glutaric acid diamide derivative is obtained as an intermediate product.

The production of the cyclic diamines which are necessary as products for I'C and I"C, respectively, takes place at the end of two reactants, one of which (in the case of the synthesis of I'C) is V"-substituted.

The circular closure is carried out according to procedures known in the literature, for example Org. Synth. 58,86 (1978),.. Macrocyclic Polyether Syntheses, Springer Verlag, Berlin, Heidelberg, New York 1982, Coord. Chem. Fox. 3.3 (1968), Ann. Chem. 1976, 916; one of the two reactants of two leaving groups and the chain end, the other two nitrogen atoms, which nucleophilically displace these leaving groups. As an example, mention should be made of the reaction of terminal dibromo, dimesyloxy, ditosyloxy or dialkoxycarbonylalkylene compounds which optionally contain one or two nitrogen atom(s) with terminal diazaalkylene compounds which optionally contain one or two additional nitrogen atom(s) in the alkylene chain, of which ( in the case of synthesis of I'c) one of the two reactants is V 1 '-substituted.

The nitrogen atoms are optionally protected, for example as tosylates, and are released before the subsequent alkylation reaction using methods known from the literature.

If diesters are used in the cyclization reaction, the diketo compounds thus obtained must be reduced using methods known to the person skilled in the art, for example with diborane.

As substituents V''', which can be transferred to V or to the substituent V" which ultimately exhibits a suitable functional group for a bond to a macro- or biomolecule, are among others hydroxy- and nitrobenzyl-, hydroxy- and carboxyalkyl-,

as well as thioalkyl radicals with up to 20 carbon atoms suitable.

They are converted according to known methods from the literature

for those skilled in the art (Chem. Pharm. Bull. 33,674 (1985), Compendium of Org. Synthesis Vol. 1-5, Wiley and Sons, Inc., Houben-Weyl,

Methoden der organischen Chemie, Volume VIII, Georg Thieme Verlag, Stuttgart, J. Biochem. 92, 1413, 1982) in the desired substituents (e.g. with the amino-, hydrazino-, hydrazinocarbonyl-, epoxide-, anhydride-, methacryloylhydrazinocarbonyl-, maleimidamidocarbonyl-, halogeno-, halogenocarbonyl-, mercapto-, isothiocyanate group as functional group), whereby in the case of the nitro-benzyl residue a catalytic hydrogenation must first be carried out (e.g. after P.N. Rylander, Catalytic Hydrogenation over Platinum Metals, Academic Press 1967).

Examples of the conversion of hydroxy or amino groups which are bound to aromatic or aliphatic residues are the reactions carried out in anhydrous, aprotic solvents such as tetrahydrofuran, dimethoxyethane or dimethylsulfoxide in the presence of an acid scavenger such as e.g. sodium hydroxide, sodium hydride or alkali or alkaline earth carbonates such as e.g. sodium, magnesium, potassium, calcium carbonate at temperatures between 0° C and the boiling point of the relevant solvent, but preferably between 20 and 60° C, with a substrate of the general fc.rmel III..

where Nf stands for a nucleofuge such as e.g. Cl, Br, J, CH-, C^H, SOo,

J b 4 o or CF^SO^, L for an aliphatic, aromatic, arylaliphatic, branched, linear or cyclic hydrocarbon residue with up to 20 carbon atoms and Fu for the desired, terminal, functional group, optionally in protected form (DE-OS 34 17,413).

As examples of compounds with the general formula III should be mentioned

The conversion of carboxy groups can, for example, be carried out according to the carbodiimide method (Fieser, Reagents for Organic Syntheses 10,142), over a mixed anhydride [Org. Prep. Pro. Int. 7,215(1975)] or over an activated ester (Adv. Org.

Chem. Part B, 4 72).

The production of the amines which are necessary as starting substances for the ring closure takes place analogously to methods known from the literature.

Starting from an N-protected amino acid, a triamine is obtained by reaction with a partially protected diamine (e.g. following the carbodiimide method), removal of the protecting groups and diborane reduction.

The reaction between a diamine obtained from amino acids (Eur. J. Med. Chem.-Chim. Ther. 21,333 (1986)) and the double molar amount of an N-protected w-amino acid gives a tetramine after suitable work-up.

In both cases, the number of carbon atoms between the N atoms can be determined using the nature of the diamines or amino acids used as coupling partners.

Some of the acid groups introduced above

the complexing units K or K in the thus obtained polymer compounds can, if desired, be further functionalized using methods known to the person skilled in the art, for example by transfer into ester, amide, hydrazide, maleimido or other groups, which are suitable for connection to bio- or macromolecules.

The thus obtained complex-forming ligands (as well as the complexes) can also be linked to bio- or macromolecules, which are known to be particularly enriched in the organ or organ part to be examined. Such molecules are, for example, enzymes, hormones, dextrans, porphyrins, bleomycins, insulin, prostaglandins, steroid hormones, amino sugars, amino acids, peptides such as polylysine, proteins (such as immunoglobulins, monoclonal antibodies, lectins) or lipids (also in the form of liposomes). Conjugates with albumins, such as human serum albumin, antibodies, such as e.g. monoclonal antibodies specific for tumor-associated antigens or antimyosin. Instead of biological macromolecules, suitable synthetic polymers such as polyethyleneimines, polyamides, polyurea, polyethers and polythiourea can also be attached. The pharmaceutical agents formed from this are, for example, suitable for use in tumor and infarction diagnostics as well as tumor therapy. Monoclonal antibodies (e.g. Nature 256, 495, 1975) have the advantages over polyclonal antibodies that they are specific for an antigenic determinant, have a defined binding affinity, are homogeneous (thus their purification becomes significantly easier) and can be produced in large amounts in cell cultures. As such are, for example, monoclonal antibodies or their fragments Fab and F(ab')2 suitable for tumor detection, which are, for example, specific for human tumors of the gastro-intestinal tract, breast, liver, bladder, gonads of melanomas (Cancer Treatment Repts. 68, 317, 1984, Bio Sei 34 , 150, 1984) or is directed against carcinoembryonic antigen (CEA), human choriogonadotropin (3-HC6) or other tumor-associated antigens, such as glycoproteins (New Engl. J. Med. 298, 1384,

1973, US-P 4,331,647). Antimyosins, anti-insulin and anti-fibrin antibodies (US-P 4,036,945) are also suitable.

Colon carcinomas can be detected NMR diagnostically using conjugates of polyethyleneimine-poly-DTPA-polyhydrazide with antibodies 17-1A (Centocor, USA) which is complexed with gadolinium(III) ions.

Conjugates or inclusion compounds with liposomes, which can for example be used as unilamellar or multilamellar phosphatidylcholine-cholesterol vesicles, are suitable for liver examinations or for tumor diagnosis, for example.

The binding of metals to the desired macro- or bio-molecules has so far taken place according to methods such as those described, for example, in Rev. Roum, Morphol. Embryol. Physio., Physiologie 1981, 18, 241 and J. Pharm. Pollock. 68, 79 (1979), for example by reaction between the nucleophilic group in a macromolecule, such as the amino, phenol, sulfhydryl, aldehyde or imidazole group and an activated derivative of the polymer complex or the ligand. As activated derivatives, for example, anhydrides, acid chlorides, mixed anhydrides (see, for example, G.E. Krejcarek and K.L. Tucker, Biochem., Biophys. Res. Commun. 1977, 581), activated esters, nitrenes or isothiocyanates come into consideration. Conversely, it is also possible to react an active macromolecule with the polymer complex or the ligand. For conjugation with proteins, there are also substituents, for example with

the structure CCH.N<*>, C.-H/,NHC0CHo, C^H.NHCS or CéH.OCH-CO. 642 64 2 64 64 2

This type of binding is, however, fraught with the disadvantage of a lack of complex stability for the conjugates or lack of specificity (eg, Diagnostic Imaging 84, 56; Science 220, 613, 1983; Cancer Drug Delivery 1, 125, 1984). The conjugate formation according to the present invention, on the other hand, takes place with the help of the functional groups found in K and/or W. In this way, up to several hundred metal ions can be bound over a binding site in the macromolecule.

In the case of antibody conjugates, the binding of the antibody to the complex or ligand must not result in a loss or reduction in binding affinity and binding specificity of the antibody to the antigen. This can either take place by binding to the carbohydrate part in the Fc part of the glycoprotein respectively in the Fab or F(ab')2 fragments or binding to the sulfur atoms

in the antibody or the antibody fragments.

In the first case, an oxidative cleavage of sugar units must first be carried out to generate coupling-capable formyl groups. This oxidation can be carried out chemically with oxidising agents such as e.g. periodic acid, sodium metaperiodate or potassium metaperiodate according to methods known from the literature (eg J. Histochem and Cytochem. 22, 1084, 1974)

in aqueous solution in concentrations from 1 to 100, preferably 1 to 20 mg/ml, and a concentration of the oxidizing agent between 0.001 to 10 mmol, preferably 1 to 10 mmol in a pH range from 4 to 8 at a temperature between 0 and 37 ° C and a reaction duration between 15 minutes and 24 hours. The oxidation can also be carried out enzymatically, for example using galactose oxidase in an enzyme concentration of 10 - 100 units/ml, a substrate concentration of 1 to 20 mg/ml, at a pH value of 5 to 8, a reaction duration of 1 to 8 hours and a temperature between 20 and 40°C (eg J. Biol. Chem. 234, 445, 1959).

Complexes or ligands with suitable functional groups such as hydrazine, hydrazide, hydroxylamine, phenylhydrazine, isemicarbazide and thiosemicarbazide are attached to the aldehydes generated by the oxidation by reaction between 0 and 37° C, with a reaction duration of 1 to 65 hours, a pH value between

5.5 and 8, an antibody concentration from 0.5 to 20 mg/ml and a molar ratio between complexing agents and antibody aldehydes of 1:1

to 1000:1. The subsequent stabilization of the conjugate takes place by reduction of the double bond, for example with sodium borohydride or sodium cyanoborohydride. The reducing agent is thereby used in an excess of 10 to 100 times (e.g. J. Biol. Chem. 254, 4359, 1979).

The second possibility for the formation of antibody conjugates is based on a gentle reduction of the disulphide bridges of immunoglobulin molecules. In this way, the more sensitive disulphide bridges between the H chains in the antibody molecule are cleaved, while the S-S binding genes in the antigen-binding region remain intact, so that in practice there is no reduction in the binding affinity and specificity of antibodies (Biochem 18. 2226, 1979, Handbook of Experimental Immunology, Vol. 1, 2nd Edition, Blackwell Scientific Publications, London 1973, Chapter 10). These free sulfhydryl groups in the inter-H chain regions are then reacted with suitable functional groups from complexing agents or metal complexes at 0 to 37° C, a pH value of 4 to 7, and a reaction duration of 3 to 72 hours under the formation of a covalent bond, which does not affect the antigen-binding region of the antibody. Examples of suitable reactive groups to be mentioned are: Halogenalkyl, haloacetyl, p-mercuribenzoate groups as well as groups that are to be subjected to a Michael addition reaction, such as e.g. maleimide, methacrylo groups (e.g.

J. Amer. Chem. Soc. 101, 3097, 1979).

For connecting the antibody fragments with the polymer complexes or In addition to the ligands, there are a number of suitable, bifunctional "connectors", which are also often partially available (see for example Pierce, Handbook and General Catalog 1986), which are reactive both towards the SH groups in the fragments and also towards amino or the hydrazine groups in the polymers.

Examples include: m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-sulfosuccinimide ester (Sulfo-MBS), N-succinimidyl-[4-(iodoacetyl)-amino]benzoic acid ester (SIAB), succinimidyl-4 (N-maleimidomethyl)-cyclohexane-1-carboxylic acid ester (SMCC),

succinimidyl-4(p-maleimidophenyl)-butyric acid ester (SMPB),

N-succinimidyl-3-(2-pyridyldithio)-propionic acid ester (SDPD), 4-[3-(2,5-dioxo-3-pyrrolinyl)-propionyloxy]-3-oxo-2,5-diphenyl-2,3 -dihydro-thiophene-1,1-dioxyd.

For the connection, bonds of a non-covalent nature can also be used, whereby ionic as well as van der Waals and hydrogen bridge bonds in alternating proportions and strength (the key-lock principle) can contribute to the bond (e.g. avidin biotin, antibody-antigen). Also inclusion compounds (host-guest) of smaller complexes in larger cavities in the macromolecule are

io possible.

The coupling principle consists in first producing a bifunctional macromolecule, whereby either an antibody hybridoma that is directed against a tumor antigen is fused with a second antibody-substance f hybridoma that is directed against a complex according to the invention, or the two other substances are joined together with each other chemically over a binder (for example in the manner indicated in J. Amer. Chem. Soc. 101, 3097 (1979) or the antibody directed against the tumor antigen, possibly over a binder, is bound to avidin (or biotin) [D.J. Hnatowich et al., J. Nucl. Med. 28. 1294 20 (1987)]. Instead of the antibodies, their corresponding F(ab) or F(ab<1>)2 fragments can also be used. For the pharmaceutical application, first the bifunctional macromolecule is injected, which is enriched at the target site, and then the complex compound according to the invention with a time interval [possibly bound to biotin (respectively 25 avidin)], which is connected in vivo at the target site and can unfold its diagnostic effect there. this can also be done by other cows bling methods come into use, such as the "Reversible Radiolabelling" described in Protein

Tailoring Food Med. Uses [Am. Chem. Soc. Symp.] (1985), 349.

so-called solid-phase coupling is a particularly simple method for producing antibody conjugates or antibody-fragment conjugates: The antibody is connected to a stationary phase (e.g. an ion exchanger), which is, for example, located in a glass column. By

successive flushing of the column with a solution suitable for

<35> generation of aldehyde groups, washing, rinsing with a solution of the functionalized complex (or the ligand), washing (if the ligand is used, another rinse is carried out with a solution containing the metal salt, followed by one more rinse) and in the end elution of the conjugate, very high conjugate yields are obtained.

This method enables an automatic and continuous production of arbitrary amounts of conjugates.

Other connection steps can also be carried out in this way.

Thus, for example, fragment conjugates can be prepared by the sequence papain reduction/bifunctional binder/functionalized complex or ligand.

The compounds thus formed are then preferably purified chromatographically over ion exchangers in a Fast-Protein-Liquid-Chromatography facility.

The production of the metal complexes according to the invention takes place in the manner described here in patent document DE-SO 34.01.052, whereby the metal oxide or a metal salt (for example the nitrate, acetate, carbonate, chloride or sulphate) of the element with the ordinal numbers 21-29, 42, 44, 57 - 83 are dissolved or suspended in water and/or a lower alcohol (such as methanol, ethanol or isopropanol) and the equivalent amount of the complexing ligand is reacted with the solution or suspension and then, if desired, replaced with acidic hydrogen atoms present in acid or . the phenol groups with cations of inorganic and/or organic bases or amino acids.

The neutralization thereby takes place with the help of inorganic bases (e.g. hydroxides, carbonates or bicarbonates) of, for example, sodium, potassium, lithium, magnesium or calcium and/or organic bases such as primary, secondary and tertiary amines, such as ethanolamine, morpholine, glucamine, N-methyl- and N,N-dimethylglucamine, as well as basic amino acids, such as for example lysine, arginine and ornithine or of amides of originally neutral or acidic amino acids.

For the preparation of the neutral complex compounds

for example, the acid complex salts in aqueous solution or suspension can be added in such a quantity of the desired bases that the neutral point is achieved. The solution obtained can then be evaporated to dryness in a vacuum. It is often advantageous to precipitate the formed neutral salts by adding solvents that are miscible with water, such as lower alcohols (methanol,

ethanol, isopropanol and others), lower ketones (acetone and others), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoxyethane and others) and thus to obtain crystallisates that are easy to isolate

and good to clean. It has been found to be particularly advantageous to add the desired base already during the complex formation in the reaction mixture and thereby save a process step.

If the acidic complex compounds contain several acidic groups, it is often appropriate to prepare neutral ones

mixed salts, which contain inorganic as well as organic cations such as counterions.

This can happen, for example, by reacting the complex-forming ligands in aqueous suspension or solution with the oxide or salt of the element that supplies the central ion and half of the amount of organic base necessary for the neutralization, the formed complex salt is isolated, the cleaned if desired and then the required amount of organic base is added for complete neutralization. The order of base addition can also be used.

Another possibility for obtaining neutral complex compounds consists in transferring the remaining acid groups in the complex in whole or in part to, for example, esters or amides. This can take place by subsequent reaction with the finished polymer complex (e.g. with exhaustive reaction of the free

carboxy groups with dimethylsulphate) which also when using

25 a suitable derivatized substrate for introducing the complexing units K-V respectively. K -V with the general formulas I'A, I'B, I'C, I'D, I"AB, I"C (e.g. N -(2,6-dioxomorpholinomethyl)-N -(ethoxy- carbonylmethyl)-3,6-diazaoctanoic acid).

The conjugates of antibody and complex are dialysed before

30 the in vivo application after incubation with a weak complexing agent, such as e.g. sodium citrate, sodium ethylenediaminetetraacetic acid, to remove weakly bound metal atoms. The polymer complexes according to the invention fulfill the many different prerequisites for being suitable as a contrast agent for nuclear spin tomography. Thus, they are eminently suitable for, after oral or parenteral application, to improve the image obtained by means of a nuclear spin tomograph in their strength by increasing the signal intensity. Furthermore, they show the great activity that is necessary to burden the body with the least possible amount of foreign substances and the good compatibility that is necessary to maintain the non-invasive nature of the examination.

The good water solubility of the agents according to the invention makes it possible to prepare highly concentrated solutions, in order to keep the volume load on the circuit within reasonable limits and compensate for the dilution with body fluid, i.e. that NMR diagnostics must be 100 - 1000 times better water soluble than for NMR spectroscopy. Furthermore, the agents according to the invention not only show a high stability in vitro, but also a surprisingly high stability in vivo, so that a release or an exchange of the non-covalently bound, in and of themselves known toxic, ions in the complexes within the time in which they new contrast agents are again excreted completely, only happening extremely slowly.

In general, the agent according to the invention is dosed for use as NMR diagnostics in amounts of 0.001 - 5 mmol metal/kg body weight, preferably 0.005 - 0.5 mmol metal/kg body weight. Details of the application are discussed, for example, in H. J. Weinmann et al., Am. J. of Roentgenology 142, 619 (1984).

Particularly lower dosages (below 1 mg/kg body weight) of organ-specific NMR diagnostics can, for example, be used to detect tumors and heart attacks.

Furthermore, the complex compounds according to the invention can advantageously be used as shift and receptivity reagents.

The agents according to the invention are likewise suitable as X-ray contrast agents, whereby it must be emphasized that they also, in comparison with the usual iodine-containing contrast agents, show a pharmacokinetics which is significantly more favorable for diagnostics. They are also particularly valuable due to the favorable absorption properties in areas with higher tube voltages for digital subtraction techniques.

In general, the agents according to the invention are dosed for use as X-ray contrast agents in analogy with, for example, meglumine diatrizoate in amounts of 0.1 - 5 mmol metal/kg body weight, preferably 0.25 - 1 mmol metal/kg body weight.

Details of the use of X-ray contrast agents are discussed, for example, in Barke, Rontgenkontrastmittel,

G. Thieme, Leipzig (1970) and P. Thurn, E. Biicheler - "Einfurung in die Rontgendiagnostik", G. Thieme, Stuttgart, New York

(1977).

The agents according to the invention are also suitable as contrast agents for ultrasound diagnostics, particularly in the form of suspensions, as their acoustic impedance is higher than the impedance of body fluids and tissues. They are generally dosed in amounts of 0.1 to

<5> 5 mmol/kg body weight, preferably of 0.25 to 1 mmol/kg body weight. Details of the application of ultrasound diagnostics are described, for example, in T.B. Tyler et al., Ultrasonic Imaging 3,323 (1981), J.I. Haft, "Clinical Echocardiography", Futura, Mount <10> Kisco, New York 1978 and G. Stefan "Echokardiograhie" G. Thieme Stuttgart/New York 1981.

In general, it has been successful to synthesize new polymer complexes, which provide new opportunities in diagnostic medicine. This development proves to be desirable above all in view of the development

i5 the development of new imaging procedures in medical diagnostics.

The polymer complexes of the general formula I' can

also used as haptens for the production of antibodies.

Details of the use of haptens for the production of anti-

<20> substance is described, for example, in S. Seil, Immunology, Immunopathology and Immunity, 372, Harper and Row Publ., 3rd ed.

The following examples serve to further explain the object of the invention.

25 Example 1

a) O-benzyl-N-trifluoroacetyltyrosine

112.5 g (0.41 mmol) of O-benzyltyrosine are suspended in

1 liter of dry methanol and added at room temperature with 5 8.9 ml

(0.42 mole) of triethylamine. After adding 67 ml (0.53 mol)

<10> trifluoroacetic acid methyl ester is stirred for 130 hours at room temperature under exclusion of water. Unreacted starting material is separated and volatile components are removed by shaking with ethyl acetate/aqueous hydrochloric acid. The ethyl acetate phase is decolorized with

activated charcoal. After evaporation of the solvent, 120.7 g are obtained

<15> (80% of theory) f heirless crystals.

Melting point: 149 - 150° C.

b) 0-benzyl-N-trifluoroacetyltyrosine-(2-carbobenzoxyamino-ethylene)-amide 18.5 g (50.4 mmol) of 0-benzyl-N-trifluoroacetyltyrosine (example 1a) are dissolved in 200 ml of dry tetrahydrofuran, add 7 ml triethylamine and then 4.8 ml (50.8 mmol) of chloroformic acid ethyl ester are added dropwise, whereby the temperature is kept below -10° C. After the addition is finished, the mixture is stirred for 30 minutes at this temperature, the same amount of pre-cooled triethylamine is added again and an ice-cold solution of 11.6 g (50.4 mmol) of N-(2-aminoethyl)-carbamic acid benzyl ester hydrochloride in 100 ml of dimethylformamide is added dropwise. It is stirred for a further 30 minutes at -10° C, then allowed to come to room temperature with stirring and then heated for 10 minutes at 30° C. The solvent is then removed on a rotary evaporator and poured into 750 ml of ice water. The crystallisate is filtered off, washed with ice water and dried. The yield amounts to 26.9 g (94% of theory). Melting point: 189 - 190° C.

c) O-benzyltyrosine-(2-carbobenzoxyaminoethylene)-amide

25.9 g (47.8 mmol) of O-benzyl-N-trifluoroacetyltyrosine-(2-carbobenzoxyamino-ethylene)-amide (Example 1b) are suspended in 300 ml of ethanol and 7.2 g (191 mmol) of sodium borohydride are added portionwise. After stirring overnight at room temperature, 50 ml of acetone is added, the solvent is removed, 500 ml of water is added and extracted several times with ethyl acetate. The organic phase gives, after drying and evaporation, 18.8 g (88% of theory) of white crystals with a melting point of 145° C.

d) Tyrosine-(2-aminoethylene)-amide

42.3 g (94.6 mmol) of the compound from example 1c

is dissolved in 1.1 liters of methanol, 2 g of 10% palladium charcoal are added and hydrogenated with stirring, until no further hydrogen uptake takes place. The catalyst is filtered off and the solvent is evaporated. It is dissolved under heat in methanol and combined

ether. 17 g (86% of theory) of colorless crystals are obtained. Melting point: 138 - 141° C.

e) 3-aza-1-(4-hydroxybenzyl)-pentane-1,5-diamine. Trihydrochloride 6.55 g (29.3 mmol) of the compound from example ld

is suspended in 130 ml of dry tetrahydrofuran and a slow stream of diborane (from 5.8 g of sodium borohydride in 75 ml of diethylene glycol-dimethylethene and 54 ml of boron trifluoride-etherate complex) with dry hydrogen is passed with constant stirring through the solution. It is stirred overnight at 60° C., then 30 ml of methanol are added dropwise at 20° C. and hydrogen chloride is introduced under ice cooling. Then boil briefly and drain. The trihydrochloride is obtained as colorless crystals (8.04 g; 86% of theory).

Melting point: 250° C (decomposition)

f) 3,6,9-triaza-4-(4-hydroxybenzyl)-3,6,9-tris-(tert-butoxy-carbonylmethyl)-undecanedioic acid bis-(tert-butyl)-diester 2.07 g (6.5 mmol) of the compound from example 1e and 5.2 g of sodium bicarbonate are placed in 60 ml of dimethylformamide (dried over sodium hydride) and added dropwise at 35° C. 6.34 g (82.2 mmol) of bromoacetic acid tert-butyl ester in 30 ml of dimethylformamide. It is stirred for a further 2.5 hours at 35° C, after which no starting product can be detected by thin-layer chromatography. Precipitated sodium bromide is filtered off and the filtrate is evaporated. The residue is added to water and extracted several times with ether. After drying and evaporation, the ether extract is purified over a silica gel column from unreacted bromoacetic acid tert-butyl ester. 3.54 g (68.8% of theory) of a colorless oil is obtained. g) 3,6,9-triaza-4-(4-benzyloxycarbonylmethoxybenzyl)-3,6,9-tris-(tert-butoxy-carbonylmethyl)-undecanedioic acid bis-(tert-butyl)-diester 1, 98 g (2.54 mmol) of the compound from example 1f are mixed with 70 mg of sodium hydride (80% in paraffin) (2.5 mmol) in 30 ml of dry tetrahydrofuran slowly with stirring and then at room temperature 0.54 g of bromoacetic acid benzyl ester (2 .54 mmol) in 20 ml of dry tetrahydrofuran. After stirring overnight, the precipitated sodium bromide is filtered off with suction, evaporated, taken up in diethyl ether and the other inorganic components are removed by washing with water. After drying over magnesium sulfate, the solvent is freed and purified over a silica gel column. 1.35 g (1.45 mmol) of a colorless syrup is obtained (62% of theory). h) 3,6,9-triaza-3,6,9-tris-(tert.-butoxycarbonylmethyl)-4-(4-carboxymethoxybenzyl)-undecanedioic acid bis-(tert.-butyl)-diester 7.83 g ( 8.43 mmol) of the compound according to example 1g is dissolved in 70 ml of dry tetrahydrofuran and hydrated in the presence of 1.4 g of 10% palladium charcoal, until no further hydrogen absorption takes place. After extraction, the solvent is removed on a rotary evaporator and the substance is dried at 0.01 torr. 4.2 g of a colorless oil are obtained (yield 74% of theory).

i) Poly-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-

aza-hexyl]-phenoxyacetyl>-polyethyleneimine

7.16 g (8.5 mmol) of the compound from example 1h are added to 80 ml of tetrahydrofuran with 1.28 g (9.4 mmol) of chloroformate isobutyl ester and 1.9 g (18.8 mmol) of triethylamine, each dissolved in 20 ml tetrahydrofuran.

After 1 hour, a solution of 533.2 mg (equivalent to 12.4 mmol mpnomer) is added while maintaining the cooling

(R)

subunit) polyethyleneimine (Polymin anhydrous) in water and allow the mixture to come to room temperature with stirring. Precipitated salt is filtered off, the solvent is evaporated and the remainder is dissolved by

heating with 150 ml of formic acid. After 3 hours at 60° C, pour into 2 liters of ice water and dialyze. After freeze-drying, 4.35 g of white, finely crystalline powder remains.

Analysis: C 51.75 H 6.03 N 10.54

j) Gadolinium complex

2.36 g of the polymeric complex former from example li is dissolved in 30 ml of water with the addition of a few drops of dilute ammonia solution and 3.92 ml of a 1 m solution of gadolinium acetate in 0.1 m ammonium acetate buffer pH 4.5 is added, whereby pH- the value when adding dilute ammonia solution is kept at a value above 5.5. It is dialysed and subjected to freeze-drying. 2.96 g of a white, crystalline powder remains.

Analysis: C 41.26 H 4.41 N 8.39 Gd 20.67

Sodium salt of gadolinium complexes

Dissolve 1.8 g of the polymer complex in 20 ml of water while stirring and adding 1N caustic soda, whereby the pH value of 7.5 must not be exceeded (consumption 4.73 ml). After freeze-drying, the sodium salt is present in the form of fine, white crystals. The yield was 1.89 g.

Analysis: C 39.0 H 3.92 N 7.94 Na 5.71 Gd 19.54

N-methylglucamine salt of the complex

2.16 g of gadolinium complex is placed in 50 ml of water and titrated with a 1 m aqueous solution of N-methylglucamine to a pH value between 7.3 and 7.4. Consumption was 6.2 ml. After freeze-drying, 3.37 g of colorless crystals are present.

Analysis: C 42.62 H 4.21 N 8.12 Gd 13.9

Example 2

Poly-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxyacetyl>-polyethyleneimine-poly-[2-(maleimido)-ethyleneamide]

3.81 g of the complex former obtained in example 1 is dissolved in 8 ml of water by adding 110 mg of potassium carbonate and quickly filled with 150 ml of dimethylsulfoxide (DMSO). The

then add 200 mg of dimethylsulphate in 1 ml of DMSO and heat for 30 minutes at 60° C. Then add 1.3 g (9.3 mmol) of 2-(maleimido)-ethylamine, heat for a further 30 minutes at 80° C and pour in three times the amount of water. After dialysis and freeze-drying, 4.0 g of white crystalline substance is present. A content of UV spectroscopically was determined

78.9 mg (0.8 mmol) maleimide/g substance (=280 mm).

Analysis: C 51.75 H 6.03 N 10.56

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 41.28 H 4.41 N 8.42 Gd 20.63

Sodium salt

C 39.02 H 3.92 N 7.96 Na 5.7 Gd 19.40

N-methylglucamine salt

C 42.63 H 4.21 N 8.14 Gd 13.88

Alternative method

If the reaction is carried out according to the above regulation, but instead of the complex former using the finished Gd complex from example lj, the derivatized complex is also obtained after analogous work-up. Of 7.85 g of

the gadolinium complex with 20.6% by weight of Gd, 7.78 g of the complex coated with amidoethylene-(maleimido) groups are obtained. The product has a gadolinium content of 20.1% by weight and 0.78 mg of maleimide per grams of substance.

Example 3

Poly-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxyacetyl>-polyethyleneimine-poly-hydrazide

As described in example 2, the hydrazide is obtained by the complexing agent, when an equivalent amount of hydrazine hydrate is added instead of 2-(maleimido)-ethylamine. The hydrazine content is determined colorimetrically (p-dimethylaminobenzaldehyde) to 22.8 mg (0.8 mmol) hydrazine/g substance. From 4.02 g of complex former, 3.87 g of hydrazide is obtained as a white, crystalline substance.

Analysis: C 51.68 H 6.07 N 10.61

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 41.21 H 4.44 N 8.46 Gd 20.65

Sodium salt

C 38.96 H 3.95 N 8.00 Na 5.70 Gd 19.52

N'— methylglucamine salt

C 42.58 H 4.23 N 8.17 Gd 13.89

Example 4

Poly-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxyacetyl>-polyethyleneimine-poly-[10-(hydrazinocarbonyl)-decylamide]

When, in the method described in example 2, 11-amino-undecanoic acid (tert.-butoxycarbonyl)-hydrazide is used as nitrogen base instead of 2-(maleimido)-ethylamine, poly-<4-[2,6-di- (bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxyacetyl>-polyethyleneimine-poly-[10-((tert-butoxycarbonyl)-hydrazinocarbonyl)decylamide from which the tert-butoxycarbonyl protecting group is removed with formic acid,

as described in example li. In this way, 6.41 g of white solid substance is obtained from 6.30 g of complex former. The hydrazine content is determined colorimetrically to be 18.7 mg (0.67 mmol) per grams of substance.

Analysis: C 51.81 H 6.07 N 10.67

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 41.38 H 4.45 N 8.52 Gd 20.52

Sodium salt

C 39.13 H 3.96 N 8.06 Na 5.67 Gd 19.40

N-methylglucamine salt

C 42.69 H 4.24 N 8.21 Gd 13.83

Example 5

a) 3,6,9-triaza-3,6,9-tris-(tert•-butoxycarbonylmethyl)-4-(4-oxiranylmethoxybenzyl)-undecanedioic acid bis-(tert-butyl)-diester

16.35 g (21.0 mmol) 3,6,9-triaza-4-(4-hydroxybenzyl)-3,6,9-tris-(tert.-butoxycarbonylmethyl)-undecanedioic acid-bis-(tert.-butyl) )-diester (example 1f) is dissolved while stirring with 630 mg (21 mmol) of sodium hydride (80% in paraffin) in 300 ml of toluene and a solution of 1.95 g (21 mmol) of epichlorohydrin in 20 ml is added dropwise at 40° C toluene. After 1 hour, carefully add 100 ml of water. After shaking, the phases separate. The organic phase is then evaporated after drying. After chromatographic purification, 15.4 g (88% of theory) of colorless oil are present.

Analysis:

b) Poly-<3-[4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxy]-2-hydroxypropyl>-polyethyleneimine

12.3 g (14.7 mmol) 3,6,9-triaza-3,6,9-tris-(tert.-butoxycarbonylmethyl)-4-(4-oxiranylmethoxybenzyl)-undecanedioic acid-bis-(tert.-butyl )-diester is dissolved in 150 ml of methanol with 6.30 mg (14.65 mmol of monomeric subunits) polyethyleneimine, whereby the temperature is kept below 5° C. During 1 hour it is heated to room temperature and it is heated for another 1 hour at 60° C.

After extraction of the solvent, heat in 100 ml of formic acid

3 hours at 60° C, poured into 2 liters of water and dialyzed. After freeze-drying, 6.22 g of a finely crystalline, white powder is present, which begins to sinter above 150° C.

Analysis: C 52.17 H 6.41 N 9.42

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 41.51 H 4.70 N 7.50 Gd 20.83

Sodium salt

C 39.22 H 4.19 N 7.08 Na 5.75 Gd 19.69

N-methylglucamine salt

C 42.79 H 4.40 N 7.52 Gd 13.97

Example 6

Poly-<3-[4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxy]-2-hydroxypropyl>-polyethyleneimine-poly-[2 --frialeimido)-ethyleneamide]

Analogous to the regulation stated for example 2, 2.9 g of the complex former obtained according to example 5 are reacted. 2.8 g of the title compound are obtained. Maleimide content

(UV) 0.31% by weight.

Analysis: C 52.17 H 6.41 N 9.45

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 41.53 H 4.70 N 7.52 Gd 20.79

Sodium salt

C 39.25 H 4.19 N 7.11 Na 5.74 Gd 19.65

N-methylglucamine salt

C 42.80 H 4.41 N 7.59 Gd 13.96

Example 7

Poly-<3-[4-[2,6-di-(bis-(carboxymethyl)-amino)- A-(carboxymethyl)-aza-hexyl]-phenoxy]-2-hydroxypropyl>-polyethyleneimine polyhydrazide

Analogous to the prescription for example 3, 3.4 g is obtained

of the hydrazide starting from 3.5 g of the complex former obtained according to example 5. The hydrazine content amounts to 14.0 mg per grams of substance.

Analysis: C 52.06 H 6.47 N 9.54

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 41.44 H 4.75 N 7.60 Gd 20.80

Sodium salt

C 39.16 H 4.24 N 7.18 Na 5.74 Gd 19.65

N-methylglucamine salt

C 42.74 H 4.44 N 7.58 Gd 13.96

Example 8

Poly-<3-[4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxy]-2-hydroxypropyl>-polyethyleneimine-poly-[10 -(hydrazinocarbonyl)-decylamide]

Analogously to the regulation stated for example 4, 4.5 g of the complex former obtained in example 5 is reacted. 4.6 g of the title compound is obtained.

Analysis: C 52.22 H 6.44 N 9.54

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 41.61 H 4.73 N 7.60 Gd 20.71

Sodium salt

C 39.33 H 4.22 N 7.18 Na 5.72 Gd 19.57

N-methylglucamine salt

C 42.85 H 4.43 N 7.59 Gd 13.92

Example 9

a) 3-aza-1-(4-hydroxybenzyl)-1,3,5-tri-tosyl-pentane-1,5-diamine 24.0 g (75.3 mmol) 3-aza-1-(4- hydroxybenzyl)-pentane-1,5-diamine (as trihydrochloride e.g. le) is added to 2 50 ml of dry pyridine and at 0° C, 37.9 g (198.7 mmol) of tosyl chloride, dissolved in 100 ml of pyridine. The mixture is allowed to stand overnight at 4° C., the main part of the pyridine is evaporated in vacuo and taken up in 300 ml of dichloromethane. By washing several times with dilute hydrochloric acid, aqueous bicarbonate solution and double-distilled water, residual pyridine and excess toluenesulfonic acid are removed. After drying, chromatograph on silica gel with ethyl acetate/hexane. The tritosylate is obtained in the form of colorless crystals. Yield 36.9 g (73% of

5 the theory).

Melting point: 145 - 146° C

0 b) 2-(4-hydroxybenzyl)-1,4,7,10-tetra-tosyl-1,4,7,10-tetra-azacyclododecane

10.95 g (16.3 mmol) of 3-aza-1-(4-hydroxybenzyl)-1,3,5-tri-tosyl-pentane-1,5-diamine are dissolved in 100 ml of dimethylformamide

and stirred with 1.35 g (45 mmol) sodium hydride (80% in paraffin)

5 for 30 minutes at 35 - 40° C. Then slowly add a solution of 9.51 g (16.3 mmol) 3-aza-1,3,5-tritosyl-1,5-dihydroxy-pentane in 100 ml DMF . It is stirred for 4 hours at 130° C, cooled overnight and the solvent distilled off. The residue is crystallized by trituration with a little methanol. After washing with pre-o diluted hydrochloric acid and recrystallization from acetonitrile is obtained

7.4 g (48% of theory) colorless crystals with melting point 192 - 193° C.

:5 c) 2-(4-hydroxybenzyl)-1,4,7,10-tetraazacyclododecane.

Trihydrobromide

12.0 g (12.7 mmol) of 2-(4-hydroxybenzyl-1,4,7,10-tetra-tosyl-1,4,7,10-tetraazacyclododecane is heated in 50 ml of glacial acetic acid

;o with 33% hydrogen bromide for 4 hours at 100° C. The mixture is poured into 300 ml of diethyl ether, filtered off with suction and resuspended twice in each time 300 ml of diethyl ether. All operations are carried out under a nitrogen protective atmosphere. After drying in vacuum available

5.16 g (78% of theory) of colorless crystals, melting at 55 115 - 117° C with decomposition.

d) 2-(4-hydroxybenzyl)-1,4,7,10-tetrakis-(tert.-butoxy-carbonylmethyl)-1,4,7,10-tetraazacyclododecane 8.49 g (16.3 mmol) 2 -(4-hydroxybenzyl)-1,4,7,10-tetraazacyclododecane (as trihydrobromide) is suspended in 150 ml of dimethylformamide, 9.66 g (115 mmol) of sodium bicarbonate are added and a solution of 12.7 g ( 65.2 mmol) bromoacetic acid tert-butyl ester in 100 ml DMF. After stirring for 2 hours at this temperature, the solvent is filtered off and the oily residue is chromatographed on silica gel with ether/hexane; A colorless viscous oil is obtained. Yield 9.0 g (79%).

e) 2-[4-(oxiranylmethoxy)-benzyl)-1,4,7,10-tetrakis-(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

12.72 g (18.2 mmol) of 2-(4-hydroxybenzyl)-1,4,7,10-tetrakis-(tert.-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (Example 9d) is dissolved in 250 ml of toluene and add 350 ml of sodium hydride (80% paraffin, 18.3 mmol). It is heated to 80 to 100° C and a solution of 1.74 g (18.2 mmol) of epichlorohydrin in 50 ml of toluene is added dropwise. After 2 hours of heating to reflux temperature, it is evaporated and purified through a silica gel column. A colorless oil is obtained.

Yield: 11.53 g (79.7% of theory)

f) Poly-<4-[2,5,8,11-tetrakis-(carboxymethyl)-2,5,8,11-tetra-azacyclododecylmethyl]-3-phenoxy-2-hydroxypropyl>-polyethyleneimine

As described in example 5, 12 g (15.1 mmol) of 2-[4-(oxiranylmethoxy)-benzyl]-1,4,7,10-tetrakis-(tert.-butoxy-carbonylmethyl)-1,4 , 7, 10-tetraazacyclododecane and 645 mg (15 mmol monomeric subunits) polyethyleneimine 7.49 g white, fine crystalline powder, which decomposes above 165°C.

Analysis: C 55.16 H 7.11 N 11.52

The gadolinium complex and its salts are obtained as described in example 1j.

Gadolinium complex

C 44.03 H 5.28 N 9.18 Gd 20.55

Sodium salt

C 42.80 H 5.01 N 8.93 Na 2.92 Gd 19.98

N-methylglucamine salt

N 44.30 H 4.99 N 8.87 Gd 16.55

Example 10

Poly-<4-[2,5,8,11-tetrakis-(carboxymethyl)-2,5,8,11-tetraaza-cyclododecylmethyl]-3-phenoxy-2-hydroxypropyl>-polyethyleneimine-poly-[2-( maleimido)-ethyleneamide]

Analogous to the regulation stated for example

2, 3.9 g of the complex former obtained according to example 9 are reacted. 4.0 g of the title compound are obtained. Maleimide content (UV): 0.48% by weight

Analysis: C 55.05 H 7.16 N 11.63

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 43.96 H 5.32 N 9.29 Gd 20.52

Sodium salt

C 42.74 H 5.04 N 9.03 Na 2.91 Gd 19.95

N-methylglucamine salt

C 44.24 H 5.03 N 8.95 Gd 16.53

Example 11

Poly-<4-[2,5,8,11-tetrakis-(carboxymethyl)-2,5,8,11-tetraaza-cyclododecylmethyl]-3-phenoxy-2-hydroxypropyl>-polyethyleneimine polyhydrazide

Analogous to the regulation stated for example 3, 2.5 g of the complex former obtained in example 9 is reacted. 2.3 g of the title compound is obtained.

Hydrazine content (colorimetric): 0.35% by weight

Analysis: C 55.18 H 7.14 N 11.61

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 42.89 H 5.31 N 9.28 Gd 20.43

Sodium salt

C 42.89 H 5.04 N 9.03 Na 2.9 Gd 19.86

N-methylglucamine salt

C 44.37 H 5.02 N 8.95 Gd 16.47

Example 12

Poly-<4-[2,5,8,11-tetrakis-(carboxymethyl)-2,5,8,11-tetraaza-cyclododecylmethyl]-3-phenoxy-2-hydroxypropyl>-polyethyleneimine-poly-[10-hydrazinocarbonyl )-decylamide]

Analogously to the regulation stated for example 4, 5.8 g of the complex former prepared in example 9 is reacted. 5.9 g of the title compound is obtained.

Hydrazine content (colorimetric): 0.35% by weight

Analysis: C 55.14 H 7.10 N 11.55

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 44.06 H 5.28 N 9.22 Gd 20.48

Sodium salt

(C 42.84 H 5.01 N 8.97 Na 2.91 Gd 19.91

N-methylglucamine salt

C 44.32 H 4.99 N 8.91 Gd 16.50

Example 13

a) N 3 -(2,6-dioxomorpholinoethyl)-N 6-(ethoxycarbonylmethyl)-3,6-diazaoctanoic acid

A suspension of 21.1 g (50 mmol) N , N -bis-(carboxymethyl)- N 9-(ethoxycarbonylmethyl)-3,6,9-triazaundecanedioic acid (J. Pharm. Sei. 68, 1979, 194) in 250 ml of acetic anhydride is stirred after the addition of 42.2 ml of pyridine for three days at room temperature. The precipitate is then filtered off, washed three times with 50 ml of acetic anhydride each time and then stirred for several hours with absolute diethyl ether. After suction, washing in absolute ethyl ether and drying in vacuum at 40° C, 18.0 g (= 89% of theory) of a white powder with a melting point of 195 - 196° C are obtained. Analysis (calculated on anhydrous substance):

b) Poly-N-f10-carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triaza-decanoyl)-polyethyleneimine

380 mg of polyethyleneimine (8.8 mmol of monomeric subunits) are dissolved in 50 ml of water. During ice-cooling, 2.37 g (5.9 mmol) of the monoanhydride obtained according to 13a are added in portions, whereby the pH value is kept at values above 9 with 1 N caustic soda. After stirring for 1 hour, dialyze and freeze-dry.

Yield 1.48 g

Analysis: C 46.55 H 6.46 H 14.51 c) The gadolinium complex and its salts are obtained analogously to example 1j.

Gadolinium complex

C 35.10 H 4.41 N 10.51 Gd 26.22

Sodium salt

C 33.25 H 4.02 N 10.33 Na 3.72 Gd 25.85

N-methylglucamine salt

C 36.95 H 4.21 N 10.02 Gd 20.40

d) The dysprosium complex and its salts are obtained as described in example 1j, when dysprosium acetate is used

instead of gadolinium acetate.

Dysprosium complex

C 34.24 H 4.25 N 10.66 Dy 26.94

Sodium salt

C 33.04 H 3.94 N 10.28 Na 3.67 Dy 25.99

N-methylglucamine salt

C 36.87 H 4.15 N 9.93 D.y 20.61

Example 14

Poly-N-[10-carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadeca-noyl)-polyethyleneimine-poly-[2-(maleimido)-ethyleneamide]

Analogous to the regulation stated for example 2, 5.2 g of the complex former prepared according to example 13 is reacted. 4.5 g of the title compound is obtained.

Maleimide content (UV): 0.27% by weight

Analysis: C 46.53 H 6.51 N 14.51

The gadolinium complex and its salts are obtained as described in Example 1 j.

Gadolinium complex

C 34.58 H 4.28 N 10.78 Gd 26.24

Sodium salt

C 33.35 H 3.95 N 10.35 Na 3.65 Gd 25.30

N-methylglucamine salt

C 37.12 H 4.15 N 10.00 Gd 20.05

Example 15

Poly-N-[10-carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadeca-noyl]-polyethyleneimine polyhydrazide

Analogous to the regulation stated for example 3, 2.5 g of the complex former prepared according to example 13 is reacted. 2.2 g of the title compound is obtained.

Hydrazine content (colorimetric): 0.47% by weight

Analysis: C 64.47 H 6.44 N 14.49

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 34.32 H 4.26 N 10.75 Gd 26.38

Sodium salt

C 33.36 H 3.96 N 10.31 Na 3.73 Gd 25.39

N-methylglucamine salt

C 37.04 H 4.15 N 9.95 Gd 20.19

Example 16

Poly-N-[10-carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadeca-noyl]-polyethyleneimine-poly-[10-(hydrazinocarbonyl)-decylamide]

Analogous to the regulation stated for example 4, 2.8 g of the complex former prepared according to example 13 are reacted. 2.9 g of the title compound are obtained.

Hydrazine content (colorimetric): 0.31% by weight

Analysis: C 46.66 H 6.43 N 14.55

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 34.44 H 4.27 N 10.72 Gd 26.20

in sodium salt

C 33.39' H 3.96 N 10.35 Na 3.72 ■ Gd 25.48

N-methylglucamine salt

C 37.07 H 4.19 N 9.96 Gd 20.00

Example 17

a) 3-aza-2-(4-benzyloxybenzyl)-4-oxoglutaric acid diamide

(Method A)

3.62 g (13.3 mmol) of O-benzyltyrosinamide is boiled with 2.7

g of ethyloxamate (23 mmol) for 14 hours in dimethoxyethane at reflux. After suctioning off the solvent, wash successively with water, ethanol and ether. After drying, 2.73 g of white crystals are obtained (60% of theory).

Melting point: 270° C.

or according to Method B.

a) 3-aza-2-(4-benzyloxybenzyl)-4-oxoglutaric acid-5-ethylester-1-amide Dissolve 3 g (11.1 mmol) of O-benzyltyrosinamide in 30 ml of dimethoxyethane, add 1.56 ml of triethylamine and add dropwise at 0° C. 1.53 g (11.1 mmol) oxalic acid ethyl ester chloride. After 30 minutes at 0° C, the mixture is poured onto 100 ml of ice, sucked off and dried. The yield was 3.87 g (94% of theory).

Melting point: 142° C.

Analysis:

P) 3.6 g (9.72 mmol) of the compound from example aoc is poured over 40 ml of a solution of 1 mol NH 2 /l methanol. After 1 hour, the precipitated product is filtered off. After drying, 3.13 g (95% of theory) of the title compound are obtained in the form of colorless crystals.

Melting point: 2 69° C

b) 3-aza-2-(4-hydroxybenzyl)-4-oxoglutaric acid diamide

lg (2.9 mmol) of the compound from example 17a is suspended with 100 mg of 10% palladium charcoal and a few drops of concentrated hydrochloric acid in 20 ml of methanol and suspended to hydrogen

recording ends. After filtration from the catalyst, 690 mg of colorless crystals are obtained (93% of theory). Melting point: 245 - 250° C (decomposition)

c) 3-aza-2-(4-hydroxybenzyl)-pentane-1,5-diamine. Trihydrochloride 1 g of the compound from example b is continued according to the regulation indicated for example le. The colorless crystallisate obtained weighed 1.19 g (93.7% of theory). Melting point: 238° C.

d) 3,6,9-triaza-3,6,9-tris-(tert-butoxycarbonylmethyl)-5-(4-hydroxybenzyl)-undecanedioic acid bis-(tert-butyl)-diester

According to the regulation indicated for example 1f, 5.19 g (16.3 mmol) of the title compound from example c is converted to 7.75 g (61% of theory) of the title compound in the form of a viscous, clear liquid.

e) 3,6,9-triaza-5-(4-benzyloxycarbonylmethoxybenzyl)-3,6,9-tris-(tert.-butoxycarbonylmethyl)-undecanedioic acid bis-(tert.-butyl)-diester 5.0 g ( 6.4 mmol) of the title compound from example d is reacted according to the instructions for example with bromoacetic acid benzyl ester to 4.6 g (74.8% of theory) of a colourless, viscous oil.

f) 3,6,9-triaza-3,6,9-tris-(tert-butoxycarbonylmethyl)-5-(4-carboxymethoxybenzyl)-undecanedoic acid bis-(tert-butyl)-diester

From 4.9 g (5.16 mmol) of the benzyl ester obtained in the preceding reaction step, 4.1 g of a colourless, viscous oil is obtained according to the procedure indicated under example lh

(93.2% of theory).

g) Poly-[4-<5-Jbis-(carboxymethyl)-aminoj-3-(carboxymethyl)-aza-2-[2-(bis-(carboxymethyl)-amino-methyl]-pentyl>-phenoxyacetyl]-polyethyleneimine

The title compound is synthesized according to the regulation indicated for example 11, from 3.27 g of the pentaester described in the previous reaction step and 245 mg of polyethyleneimine. In this way, 2.0 g of the polymeric complex-former is obtained as a white, crystalline powder.

Analysis: C 51.76 H 6.04 N 10.56

The gadolinium complex and its salts are obtained as described in Example 1 j.

G adolinium complex

C 41.32 H 4.42 N 8.45 Gd 20.70

Sodium salt

C 38.91 H 3.91 N 7.94 Na 5.71 Gd 19.58

N-methylglucamine salt

C 42.60 H 4.23 N 8.13 Gd 13.87

Example 18

Poly-[4-<5-{bis-(carboxymethyl)-amino}-3-(carboxymethyl)-aza-2-[2-(bis-(carboxymethyl)-amino)-methyl]-pentyl>-phenoxyacetyl]- polyethyleneimine-poly-[2-(maleimido)-ethyleneamide]

Analogous to the regulation stated for example 2, 4.3 g of the complex former obtained from example 17 is reacted. 4.1 g of the title compound is obtained. Maleimide content (UV) 0.68% by weight.

Analysis: C 51.74 H 6.05 N 10.55

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 41.09 H 4.43 N 8.43 Gd 20.69

Sodium salt

C 38.95 H 3.93 N 7.98 Na 5.71 Gd 19.50

N-methylglucamine salt

C 42.54 H 4.21 N 8.15 Gd 13.88

Example 19

Poly-[4-<5-{bis-(carboxymethyl)-amino}-3-(carboxymethyl)-aza-2-[2-(bis-(carboxymethyl)-amino)-methyl]-pentyl>-phenoxyacetyl]- polyethyleneimine polyhydrazide

Analogously to the regulation stated for example 3, 2.6 g of the complex former obtained according to example 17 is reacted. 2.5 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.32% by weight

Analysis: C 51.91 H 6.05 N 10.52

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 41.06 H 4.41 N 8.43 Gd 20.71

Sodium salt

C 39.13 H 3.93 N 7.93 Na 5.73 Gd 19.48

N-methylglucamine salt

C 42.57 H 4.21 N 8.11 Gd 13.92

Example 20

Poly-[4-<5-{bis-(carboxymethyl)-aminoj -3-(carboxymethyl)-aza-2-[2-(bis-(carboxymethyl)-amino)-methyl]-pentyl>-phenoxyacetyl]-polyethyleneimine -poly-[10-(hydrazinocarbonyl)-decylamide]

Analogously to the regulation stated for example 4, 2.8 g of the complex former obtained according to example 17 is reacted. 2.9 g of the title compound is obtained. Hydrazine content (colorimetric) 0.77% by weight

Analysis: C 52.03 H 6.09 N 10.65

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 41.29 H 4.47 N 8.52 Gd 20.52

Sodium salt

C 39.14 H 3.96 N 8.06 Na 5.67 Gd 19.40

N-methylglucamine salt

C 42.76 H 4.24 N 8.17 Gd 13.79

Example 21

a) 3,6-diaza-3,6-bis-(tert-butoxycarbonylmethyl)-4-(4-hydroxy-benzyl)-suberic acid bis-(tert-butyl)-diester. 15.31 g (0.064 mol) of 4-hydroxybenzyl-1,2-ethanediamine as dihydrochloride and 71.14 g (0.71 mol) of potassium bicarbonate are added to 380 ml of dimethylformamide (dried over sodium hydride) and added dropwise at 35° C 50 g (0.26 mol) of bromoacetic acid tert-butyl ester in 80 ml of dimethylformamide. It is stirred for a further 2.5 hours at 35° C, after which no starting product can be detected by thin-layer chromatography. Precipitated potassium bromide is filtered off and the filtrate is evaporated. The residue is added to water and extracted several times with ether. After drying and evaporation, the ether extract is purified through a silica gel column for unreacted bromoacetic acid tert-butyl ester. 24.8 g (63% of theory) of a colorless oil is obtained.

b) Poly-[4-<2,3-di-(bis-(carboxymethyl)]-aminopropyl>-phenyl-iminocarbarnate]-polyethyleneimine

7.2 g (11.56 mmol) of the title compound from a) are dissolved in 150 ml of methanol and 1.4 g (13.3 mmol) of cyanogen bromide are added in portions, whereby an equimolar amount of 0.1 n methanolic potassium hydroxide is simultaneously added dropwise . The temperature must therefore not exceed 10° C. After 15 minutes, a solution of

470 mg (10.9 mmol of monomeric subunits) polyethyleneimine in 20 ml of methanol and slowly heated to 40° C. After half an hour at this temperature, the solvent is evaporated (after any precipitate has been filtered off beforehand) and the residue is dissolved in 150 ml of formic acid during heating. After 2 hours at 60° C, the mixture is poured into 2 liters of ice water, dialyzed and the retentate subjected to freeze drying. The white, fluffy crystallite begins to decompose above 80° C. The yield was 3.56 g.

Analysis: C 51.52 H 5.65 N 12.13

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 38.59 H 3.77 N 9.14 Gd 25.10

Sodium salt

C 37.45 H 3.47 N 8.80 Na 3.57 Gd 24.37

N-methylglucamine salt

C 40.14 H 3.78 N 8.76 Gd 19.46

Example 22

Poly-[4-<2,3-di-(bis-(carboxymethyl))-aminopropyl]-phenylimino-carbamate>-polyethyleneimine-poly-[2-(maleimido)-ethylamide]

Analogous to the regulation stated for example 2, 2.5 g of the complex former obtained in example 21 is reacted. 2.2 g of the title compound is obtained.

Maleimide content (UV) 0.67% by weight

Analysis: C 51.46 H 5.66 N 12.18

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 38.85 H 3.77 N 9.10 Gd 25.17

Sodium salt

C 37.63 H 3.48 N 8.80 Na 3.55 Gd 24.42

N-methylglucamine salt

C 40.36 H 3.79 N 8.80 Gd 19.50

Example 23

Poly-[4-<2,3-di-(bis-(carboxymethyl))-aminopropyl]-phenylimino-carbamate>-polyethyleneimine polyhydrazide

Analogously to the regulation stated for example 3, 4.4 g of the complex former obtained in example 21 is reacted. 4.2 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.32% by weight

Analysis: C 51.64 H 5.66 N 12.08

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 38.79 H 3.77 N 9.09 Gd 25.21

Sodium salt

C 37.57 H 3.48 N 8.77 Na 3.54 Gd 24.44

N-meglumine salt

C 40.41 H 3.79 N 8.80 Gd 19.48

Example 24

Poly-[4-<2,3-di-(bis-(carboxymethyl))-aminopropyl]-phenylimino-carbamate>-polyethyleneimine-poly-[10-(hydrazinocarbonyl)-decylamide]

Analogous to the regulation stated for example 4, 3.1 g of the complex former obtained in example 21 is reacted. 3.2 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.17% by weight

Analysis: C 51.43 H 5.70 N 12.16

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 39.03 H 3.81 N 9.24 Gd 25.02

Sodium salt

C 37.41 H 3.51 N 8.93 Na 3.53 Gd 24.35

N-meglumine salt

C 40.56 H 3.82 N 8.81 Gd 19.40

Example 25

a) N-carbobenzoxyserine-(2-carbobenzoxyaminoethylene)-amide

7.34 g (30.7 mmol) of N-carbobenzoxyserine are dissolved in

120 ml of dry tetrahydrofuran, 5 ml of Et^N and then 2.9 ml of chloroformic acid ethyl ester are added dropwise, whereby the temperature is kept below -10° C. After the addition is finished, the mixture is stirred for 30 minutes at this temperature, the same amount of pre-cooled triethylamine is added again and an ice-cold solution of 7.1 g

(30.7 mmol) of N-(2-aminoethyl)-carbamic acid benzyl ester hydrochloride in 70 ml of dimethylformamide is added dropwise. It is stirred for a further 30 minutes at -10° C, the mixture is then allowed to come to room temperature with stirring and then heated for 10 minutes to 30° C. The solvent is then removed on a rotary evaporator and poured into 500 ml of ice water. The crystallisate is filtered off, washed with ice water and dried. The yield was 10.33 g (81% of theory). Melting point: 167° C.

b) (2-aminoethyl)-serinamide

13.46 g (32.4 mmol) of N-carbobenzoxyserine-(2-carbobenz-oxyaminoethylene)-amide is hydrogenated in 200 ml of methanol in the presence of 1.37 g of 10% palladium/charcoal until no more hydrogen is absorbed. The catalyst is filtered off and all volatile parts are removed using an oil pump. A tough, partially crystalline oil remains.

Yield: 4.67 g (98% of theory)

c) 1-hydroxymethyl-1,3,5-triazapentane. Trihydrochloride.

4.3 g (29.3 mmol) (2-aminoethyl)-serinamide (Ex

25b) is suspended in 130 ml of dry tetrahydrofuran and a slow stream of I^Hg (from 5.6 g of NaBH^ in 75 ml of diethylene glycol dimethyl ether and 54 ml of boron trifluoride etherate complex) is run with dry

nitrogen with constant stirring through the solution. It is stirred overnight at 60° C., then 30 ml of methanol are added dropwise at 20° C. and hydrogen chloride is introduced under ice cooling. It is then boiled for a short time and suctioned off. The trihydrochloride is obtained as a white, crystalline powder in a 69% yield.

Melting point: 236° C (decomposition)

d) 3,6,9-triaza-3,6,9-tris-(tert.-butoxycarbonylmethyl)-4-hydroxymethyl-undecanedioic acid bis-(tert.-butyl)-diester 17.85 g (73.59 mmol ) of the triamine obtained according to 25c), is added to 450 ml of dimethylformamide and 54.4 g (648 mmol) of sodium bicarbonate are added. At 35° C, 78.95 g (404.72 mmol) of bromoacetic acid tert-butyl ester are added dropwise while stirring, then stirred for a further 3 hours at 35° C and the precipitated salt is filtered off. After evaporation, water is added to the residue and extracted several times with ether. Unreacted bromo-acetic acid ester is separated on a silica gel column and, after suctioning off the solvent, 42.32 g (6 0.12 mmol) of a colorless, viscous oil (81.7% of theory) is obtained e) 3,6,9-triaza- 3,6,9-tris-(tert.-butoxycarbonylmethyl)-4-[(oxiranylmethoxy)-methyl]-undecanedic acid bis-(tert.-butyl)-diester 24.16 g (34.32 mmol) of the hydroxymethyl -compound which is obtained in 25d), dissolve in 500 ml of dry toluene with 1.47 g (37.76 mmol) of sodium amide and add at 40° C slowly a solution of 3.46 g (37.41 mmol) of epichlorohydrin in 50 ml toluene. After one hour of stirring at this temperature, insoluble substances are sucked off, evaporated and purified through a silica gel column. 24.0 g (31.58 mmol) of colorless oil remain (92% of theory).

f) Poly-<6,10-di-[bis-(carboxymethyl)-amino]-8-[(carboxymethyl)-aza]-2-hydroxy-4-oxa-decyl>-polyethyleneamine

As described in Example 5, the title compound is obtained from 4.2 g of the compound obtained in the previous reaction step, and 230 mg of polyethyleneimine as a colorless, crystalline powder in a yield of 2.31 g. The compound sinters above 145° C. with gradual darkening .

Analysis: C 45.99 H 6.57 N 10.77

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 35.51 H 4.63 N 8.32 Gd 23.18

Sodium salt

C 33.28 H 4.06 N 7.08 Na 6.34 Gd 21.67

N-meglumine salt

C 39.08 H 4.32 N 8.08 Gd 15.05

Example 2 6

Poly-<6,10-di-[bis-(carboxymethyl)-amino]-8-[(carboxymethyl)-aza]-2-hydroxy-4-oxa-decyl>-polyethyleneimine-[2-(maleimido)-ethyleneamide ]

Analogous to the regulation stated for example 2, 2.5 g of the complex former obtained in example 25 is reacted. 2.4 g of the title compound is obtained.

Maleimide content (UV) 0.30% by weight

Analysis: C 46.17 H 6.54 N 10.8

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 35.43 H 4.65 N 8.41 Gd 23.20

Sodium salt

C 33.45 H 4.09 N 7.83 Na 6.31 Gd 21.77

N-meglumine salt

C 39.23 H 4.35 N 8.10 Gd 14.90

Example 27

Poly-<6,10-di-[bis-(carboxymethyl)-amino]-8-[(carboxymethyl)-aza]-2-hydroxy-4-oxa-decyl>-polyethyleneimine polyhydrazide

Analogous to the regulation stated for example 3, 2.3 g of the complex former obtained according to example 25 is reacted. 2.3 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.37% by weight

Analysis: C 45.89 H 6.59 N 10.75

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 35.66 H 4.64 N 8.29 Gd 23.11

Sodium salt

C 33.42 H 4.05 N 7.82 Na 6.35 Gd 21.7

N-meglumine salt

C 39.12 H 4.32 N 8.01 Gd 15.00

Example 2 8

Poly-<6,10-di-[bis-(carboxymethyl)-amino]-8-[(carboxymethyl)-aza]-2-hydroxy-4-oxa-decyl>-polyethyleneimine-poly-[10-(hydrazinocarbonyl) -decylamide]

Analogously to the regulation stated for example 4, 3.0 g of the complex former obtained according to example 25 is reacted. 3.1 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.46% by weight

Analysis: C 46.06 H 6.61 N 10.81

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 35.74 H 4.66 N 8.39 Gd 23.05

Sodium salt

C 33:.32 H 4.08 N 7.90 Na 6.33 Gd 21.6

N-meglumine salt

C 39.25 H 4.33 N 8.06 Gd 14.99

Example 29

Poly-[4-<2,6-di-(bis-(carboxymethyl)-amino]-4-(carboxymethyl)-aza-hexyl]-phenoxyacetyl>-polylysine

To a solution of 3.21 g (25 mmol of lysyl subunits) polylysine and 2.8 g of KOH in 150 ml of water is added dropwise at 0° C a solution prepared from 25.14 g (30 mmol) of the title compound from Example 1h, 4.7 g (30 mmol) chloroformic acid isobutyl ester and 3.03 g (30 mmol) triethylamine in 100 ml tetrahydrofuran at 0° C. After the addition is finished, the precipitate is decanted and this is washed with water. The precipitate is dissolved in 250 ml of hot formic acid and then heated for 2 hours to 50° C. The mixture is then added to 3 liters of water, it is dialysed and the retentate is subjected to freeze drying. Fine white crystals are obtained in a yield of 9.07 g.

Analysis: C 52.25 H 6.15 N 10.63

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 42.51 H 4.65 N 8.68 Gd 19.04

Sodium salt

C 40.55 H 4.20 N 8.20 Na 5.28 Gd 18.07

N-meglumine salt

C 43.63 H 4.40 N 8.34 Gd 13.16

Example 30

Poly-[4-<2,6-di-(bis-(carboxymethyl)-amino]-4-(carboxymethyl)-aza-hexyl]-phenoxyacetyl>-polylysine-poly-[2-(maleimido)-ethyleneamide]

Analogously to the regulation stated for example 2, 4.3 g of the complex former obtained according to example 29 is reacted. 4.1 g of the title compound is obtained.

Maleimide content (UV) 0.35% by weight

Analysis: C 52.41 H 6.16 N 10.64

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 44.42 H 4.66 N 8.68 Gd 18.84

Sodium salt

C 40.60 H 4.17 N 8.28 Na 5.25 Gd 17.95

N-meglumine salt

C 43.23 H 4.38 N 8.33 Gd 13.01

Example 31

Poly-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxyacetyl>-polylysine-poly-hydrazide

Analogous to the regulation stated for example 3, 4.6 g of the complex former obtained in example 29 is reacted. 4.5 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.52% by weight

Analysis: C 52.29 H 6.13 N 10.63

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 42.47 H 4.64 N 8.67 Gd 19.03

Sodium salt

C 40.34 H 4.20 N 8.21 Na 5.25 Gd 17.93

N-meglumine salt

C 43.59 H 4.37 N 8.32 Gd 13.14

Example 32

Poly-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl)-phenoxyacetyl>-polylysine-[10-(hydrazinocarboxyl)-decylamide]

Analogous to the regulation stated for example 4, 5.3 g of the complex former obtained according to example 29 is reacted. 5.4 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.31% by weight

Analysis: C 52 , 3 H 6 , 17 N 10 , 69

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 42.77 H 4.65 N 8.70 Gd 18.84

Sodium salt

C 40.41 H 4.18 N 8.29 Na 5.24 Gd 17.87

N-meglumine salt

C 43.34 H 4.40 N 8.33 Gd 13.09

Example 33

Poly- {3-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxy>-2-hydroxypropylJ-polylysine

A solution of 8.1 g (14.58 mmol) of 3,6,9- triaza-3,6,9-tris-(carboxymethyl)-4-[4-(oxiranylmethoxy)-benzyl]-undecanedic acid (prepared from the corresponding penta-(tert-butyl ester) from example 5a by heating with formic acid corresponding to the prescription in example li) in 150 ml of 0.1 N caustic soda. The mixture is heated to room temperature, dialyzed and the retentate subjected to freeze drying. 8.36 g of colorless substance in the form of fine needles is obtained.

Analysis: C 55.27 H 7.14 N 11.62

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 45.01 H 5.43 N 9.41 Gd 19.26

Sodium salt

C 43.80 H 5.17 N 9.18 Na 2.74 Gd 18.81

N-meglumine salt

C 44.79 H 5.14 N 4.05 Gd 15.75

Example 34

Poly- {3-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxy>-2-hydroxypropyl}-polylysine-poly-[2 -(maleimido)-ethyleneamide]

Analogous to the regulation stated for example

2, 5.3 g of the complex former obtained according to example 33 are reacted. 5.1 g of the title compound are obtained.

Maleimide content (UV) 0.44% by weight

Analysis: C 55.43 H 7.11 N 25.00

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 45.02 H 5.43 N 9.43 Gd 19.14

Sodium salt

C 43.85 H 5.14 N 9.16 Na 2.74 Gd 18.73

N-meglumine salt

C 44.92 H 5.13 N 9.08 Gd 15.65

Example 35

Poly-{3-<4-[2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxy>-2-hydroxypropylj-polylysine-poly-hydrazide Analogous to the regulation indicated for example 3, 4.7 g of the complex former obtained according to example 33 are reacted. 4.6 g of the title compound are obtained.

Hydrazine content (colorimetric) 0.12% by weight

Analysis: C 55.19 H 7.12 N 11.59

The gadolinium complex and its salts are obtained as described in Example 1j.

Gadolinium complex

C 44.77 H 5.40 N 9.43 Gd 19.17

Sodium salt

C 43.83 H 5.14 N 9.20 Na 2.75 Gd 18.67

N-meglumine salt

C 45.08 H 5.11 N 9.10 Gd 15.66

Example 3 6

Poly-(3-<4-[ 2 , 6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza-hexyl]-phenoxy>-2-hydroxypropyl}-polylysine-poly-[10 -(hydrazinocarbonyl)-decylamide]

Analogous to the regulation stated for example 4, 3.8 g of the complex former obtained in example 33 is reacted. 3.9 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.14% by weight

Analysis: C 55.54 H 7.18 N 11.64

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 45.00 H 5.46 N 9.46 Gd 19.08

Sodium salt

C 43.82 H 5.16 N 9.20 Na 2.73 Gd 18.64

N-meglumine salt

C 45.17 H 5.14 N 9.10 Gd 15.69

Example 37

Poly-N-[10-carboxy-3,6,9-tris-(carboxymethyl)-3,6,9-triaza-decanoyl]-poly-L-lysine

Dissolve 632 mg (4.9 mmol of monomeric subunits) of polylysine in 50 ml of water with 5 ml of 1 N caustic soda and add under

ice cooling portions 2.1 g (5.2 mmol) N 3-(2,6-dioxomorpholino-ethyl)-N -(ethoxycarbonylmethyl)-3,6-diazaoctanoic acid (Example 13a), whereby the pH value is maintained at above 9 with 1 n caustic soda. The mixture is stirred overnight while warming to room temperature and dialyzed. After freeze-drying, 2.05 g of colorless, crystalline needles are present.

Analysis: C 48.06 H 6.61 N 14.21

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 36.99 H 4.66 N 10.91 Gd 23.34

Sodium salt

C 35.80 H 4.35 N 10.63 Na 3.27 Gd 22.44

N-meglumine salt

C 38.78 H 4.46 N 10.19 Gd 18.20

Example 38

Poly-N g -[10-carboxy-3,6,9-tris-(carboxymethyl)-3,6,9-triaza-decanoyl]-poly-L-lysine-poly-[2-(maleimido)-ethyleneamide]

Analogous to the regulation stated for example 2, 2.7 g of the complex former obtained according to example 37 is reacted. 2.4 g of the title compound is obtained.

Maleimide content (UV) 0.88% by weight

Analysis: C 48.10 H 6.61 N 14.26

The gadolinium complex and its salts are obtained as described in Example lj.

Gadolinium complex

C 37.12 H 4.64 N 11.02 Gd 23.14

Sodium salt

C 35.87 H 4.36 N 10.62 Na 3.29 Gd 22.99

N-meglumine salt

C 38.83 H 4.49 N 10.28 Gd 18.15

Example 39

a) Poly-N-[10-carboxy-3,6-bis-(carboxymethyl)-9-ethoxy-carbonylmethyl-3,6,9-triazadecanoyl]-poly-L-lysine

0.82 g (5 mmol) of poly-L-lysine hydrochloride is dissolved in 100 ml of water. While maintaining a pH value of 9.5, 6.06 g (15 mmol) N 3-(2,6-dioxomorpholinoethyl).-N<6->(ethoxycarbonylmethyl)-3,6-diazaoctanoic acid are added in portions (Example 13a ), is adjusted to pH 7 with approx. 11 ml of 1 N hydrochloric acid and desalted as an ultrafiltration membrane CAmicon" YM2) and then freeze-dried. Yield: 2.6 g (90% of theory).

Ethoxy determination: 6.85%, this corresponds to an acylation of the poly-lysine of 88%.

Melting point: 247° C (decomposition)

3+

1 g of this compound complexes 240 mg of Gd

b) Poly-N<6->[10-carboxy-3,6,9-tris-(carboxymethyl)-3,6,9-triazadecanoyl]-poly-L-lysine polyhydrazide

2.4 g (4.2 mmol) of the ethyl ester described

in example 39a, dissolve in 100 ml of water and, after adding 5 ml (0.5 mmol) of 0.1 M hydrazine hydrate solution, stir for 4 hours at reflux and overnight at room temperature. At a pH value above 9, the solution is ultrafiltered, the residual solution is adjusted to a pH value of 4 after adding "Amberlite" IR 120(H<+>)

and freeze-dried.

Yield: 2 g

Hydrazide content: 0.3 mol%

c) Gadolinium complex of

Poly-N<6->[10-carboxy-3,6,9-tris-(carboxymethyl)-3,6,9-triaza-decanoyl]-poly-L-lysine polyhydrazide

Dissolve 1.9 g of the complexing agent described in example 39b in 200 ml of water, add 548 mg Gd»0, = 475 mg Gd 3~h and stir for 1 hour at 80 o C. The resulting solution is ultrafiltered and freeze-dried thereafter.

Yield: 2.35 g

Gd content: 20% by weight

Åmax (H20) = 201 nm (e = 9000)

The following relaxivities were measured [the measurements of the relaxation times Tl and T2 take place in a "Minispec" p 20

(User) at 0.46 Tesla (= 20 MHz), 37° C]:

T-^-relaxivity: 11.38 (L/mmol sec)

T2 reactivity: 13.13 (L/mmol sec)

Example 40

Poly-N<6->[10-carboxy-3,6,9-tris-(carboxymethyl)-3,6,9-triaza-decanoyl]-poly-L-lysine-poly-[10-(hydrazino-carbonyl )-decylamide]

Analogous to the regulation stated for example 4, 2.9 g of the complex former prepared according to example 37 is reacted. 3.0 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.41% by weight

Analysis: C 48.3 H 6.60 N 14.23

Gadolinium complex

C 37.10 H 4.68 N 10.96 Gd 23.31

Sodium salt

C 35.84 H 4.39 N 10.67 Na 3.28 Gd 22.49

N-meglumine salt

C 38.87 H 4.48 N 10.29 Gd 18.27

Example 41

a) 3,6,9-triaza-3,6,9-tris-(tert-butoxycarbonylmethyl)-4-[4-(3-benzyloxycarbonylaminopropoxy)-benzyl)]-undecanedioic acid-bis-(tert-butyl) - diesters

4.6 g (5.90 mmol) of the compound according to example 1f are mixed with 194 mg of NaH (80% in paraffin) (6.48 mmol)

in 40 ml of dry tetrahydrofuran and to this slowly add 1.6 g of N-(3-bromopropyl)-carbamic acid benzyl ester in 20 ml of tetrahydrofuran. After stirring overnight, it is evaporated and separated from paraffin oil through a silica gel column. After evaporation of the solvent, 4.2 g of a colorless oil is obtained (yield 74% of theory).

b) 3,6,9-triaza-3,6,9-tris-(tert•-butoxycarbonylmethyl)-4-[4-(3-aminopropoxy)-benzyl]-undecanedioic acid-bis-(tert-butyl)- diester 3.9 g (4.8 mmol) 3,6,9-triaza-3,6,9-tris-(tert.-butoxycarbonylmethyl)-4-[4-(3-benzyloxycarbonylaminopropoxy)-benzyl)]-undecanedioic acid -bis-(tert-butyl)-diester (Example 41a) is dissolved in 100 ml of methanol and hydrogenated with 2.13 g of 10% palladium charcoal> until no more H- is taken up. The catalyst is then filtered off. The remaining colorless oil weighed 3.17 g (97.3% of theory).

c) Polyacrylpoly-<3-(4-|2,6-di-(bis-(carboxymethyl)-amino)-4-((carboxymethyl)-aza)-hexyl]-phenoxyj-propyl>-amide

To a solution of 10.1 g of polyacrylic acid polyethylene ester in 100 ml of toluene, a solution of 83.7 g (100 mmol) of the compound produced in the previous reaction step (41b) in 100 ml of toluene is added dropwise at 60 - 80° C and kept for 20 hours at this temperature. The solvent is filtered off and heated for 10 hours with 1 liter of trifluoroacetic acid. After dilution with 10 liters of water, it is dialysed and freeze-dried in portions. A total of 45.46 g of a colourless, fibrous crystalline polymer is obtained.

Analysis: C 51.08 H 6.69 N 9.45

Gadolinium complex

C 40.69 H 4.91 N 7.47 Gd 20.97

Sodium salt

C 38.17 H 4.36 N 7.07 Na 5.81 Gd 19.94

N-meglumine salt

C 42.13 H 4.55 N 7.52 Gd 14.05

Example 42

Polyac.ry.lpoly-<3-|4-H,2 , 6-di-(bis-(carboxymethyl)-amino)-4-((carboxymethyl)-aza)-hexylyl-phenoxyj-propyl>-amide- poly-H.2-(maleimido)-ethylamidn

Analogously to the regulation stated for example 2, 6.2 g of the complex former prepared according to example 41 is reacted. 6.0 g of the title compound is obtained.

Maleimide content (UV) 0.44% by weight

Analysis: C 51.22 H 6.7 N 9.45

Gadolinium complex

C 40.61 H 4.91 N 7.54 Gd 20.99

Sodium salt

C 38.36 H 4.36; N 7.10 Na 5.83 Gd 19.88

N-meglumine salt

C 42.35 H 4.54 N 7.53 Gd 14.04

Example 43

PolyaQrylpoly-<3-|4-t 2 , 6-di-(bis-(carboxymethyl)-amino)-4-((carboxymethyl)-aza)-hexyl]-phenoxyj-propyl>-amide-poly-[10- (hydrazinocarbonyl)-decylamide]

Analogous to the regulation stated for example 4, 4.3 g of the complex former prepared according to example 41 is reacted. 4.4 g of the title compound is obtained.

Hydrazine content (colorimetric) 0.56% by weight

Analysis: C 51.26 H 6.73 N 9.54

Gadolinium complex

C 40.61 H 4.94 N 7.58 Gd 20.91

Sodium salt

C 38.35 H 4.37 N 7.15 Na 5.81 Gd 19.76

N-meglumine salt

C 42.17 H 4.54 N 7.57 Gd 14.07

Example 44

Polyacrylpoly-<3-|4-[ 2,6-di-(bis-(carboxymethyl)-amino)-4-((carboxymethyl)-aza)-hexyl]-phenoxyj-propyl>-amide poly-hydrazide

A solution of 3.5 g of polyacrylic acid polyethylene ester i

To 500 ml of toluene is added a solution of 29 g (34 mmol) of the title compound from example 41a and 5 ml of a tetrahydrofuran solution, which contains 150 mg of hydrazine per litres, simultaneously at room temperature. It is slowly heated to 80° C and the procedure is as described in example 41c.

Yield: 15.39 g (colorless crystals)

Analysis: C 50.90 H 6.95 N 9.43

Gadolinium complex

C 40.38 H 4.88 N 7.49 Gd 21.04

Sodium salt

C 38.43 H 4.37 N 7.10 Na 5.81 Gd 19.87

N-meglumine salt

C 41.94 H 4.53 N 7.50 Gd 14.12

Example 45

a) 3,J>,9-triaza-3,6,9-tris-(tert-butoxycarbonylmethyl)-4-[3-(benzyloxycarbonyl)-aminopropoxymethyl]-undecanedoic acid bis-(tert-butyl)-diester

Starting from 3,6,9-triaza-3,6,9-tris-(tert.-butoxy-carbonylmethyl)-4-hydroxymethyl-undecanedioic acid bis-(tert.-butyl)-diester (Example 25d) the title compound is obtained analogous to the regulation for example 41a in 71% yield.

b) 3,6,9-triaza-3,6,9-tris-(tert-butoxycarbonylmethyl)-4-[3-aminopropoxymethyl]-undecanedoic acid bis-(tert-butyl)-diester

Starting from the benzyloxycarbonyl-protected amino compound that was obtained in the previous reaction step (example 45a), the title compound is obtained analogously to the procedure indicated for example 41b in 93% yield.

c) Polyethyleneimine polyacetic acid polymethyl ester

8.18 g polyethyleneimine (186.5 mmol monomeric units),

20.1 g of triethylamine (200 mmol) and 30 g of bromoacetic acid methyl ester (196 mmol) are mixed under ice-cooling in 250 ml of methanol and heated overnight to room temperature. The solvent is removed and it is extracted several times with hot methylene chloride. After drying, 19 g of oil remains.

Analysis: C 60.03 H 9.13 N 14.14

d) Polyethyleneimine polyacetic acid poly-<3- | [2,6-di-(bis-(carboxymethyl)-amino)-4-(carboxymethyl)-aza)-hexyl]-methoxyj-propyl>-amide

3.65 g of polyethyleneimine polyacetic acid polymethyl ester (example 45c) is dissolved in 50 ml of methanol and a solution of 31 g (42 mmol) of the title compound from example 45b is added. It is heated for 10 hours to 60° C, the solvent is suctioned off and the ester is cleaved with formic acid as described in example li. After dialysis and freeze-drying, 15.07 g of the complexing agent are present in the form of fine, crystalline needles.

Analysis: C 43.24 H 7.07 N 13.28

Gadolinium complex

C 33.66 H 5.04 N 10.27 Gd 22.86

Sodium salt

C 31.40 H 4.44 N 9.64 Na 6.29 Gd 21.59

N-meglumine salt

C 37.51 H 4.61 N 9.34 Gd 14.96

Example 46

i Polyethyleneimine polyacetic acid poly-<3-|[2,6-di-(bis-(carboxymethyl)-amino)-4-((carboxymethyl)-aza)-hexyl]-oxyj-propyl>-amide polyhydrazide

3.12 g of polyethyleneimine polyacetic acid polymethyl ester

(example 45c) is dissolved in 100 ml of methanol and 1 ml of a solution containing 50 mg of hydrazine hydrate per 100 ml.

It is heated for 30 minutes to 40° C. and then 26 g (35.2 mmol) of the title compound from example 45b dissolved in 50 ml of methanol are added. The further processing takes place as described for example 41c. 12.17 g of fine crystalline substance is obtained.

Hydrazine content: 0.13% by weight

Analysis: C 43.35 H 7.10 N 13.25

Gadolinium complex

C 33.45 H 5.04 N 10.31 Gd 22.97

Sodium salt

C 31.52 H 4.45 N 9.68 Na 6.28 Gd 21.61

N-meglumine salt

C 37.62 H 4.61 N 9.30 Gd 14.91

Example 47

Polyethyleneimine polyacetic acid poly-<3-|[2,6-di-(bis-(carboxymethyl)-amino)-4-((carboxymethyl)-aza)-hexyl]-oxyj-propyl>-amide-poly-[2-(maleimido )-ethylamide]

If hydrazine in example 46 is replaced by an equivalent amount of 2-(maleimido)-ethylamine (which is added in the form of its trifluoroacetate), the corresponding polymer with the maleimide function is obtained.

Maleimide content (UV): 0.38% by weight

Analysis: C 43.05 H 7.10 N 13.31 Gd 36.46

Gadolinium complex

C 33.45 H 5.05 N 10.28 Gd 22.95

Sodium salt

C 31.52 H 4.46 N 9.64 Na 6.30 Gd 21.56

N-meglumine salt

C 37.79 H 4.61 N 9.33 Gd 14.90

Example 48

Polyethyleneimine polyacetic acid poly-<3-|[2,6-di-(bis-(carboxymethyl)-amino)-4-((carboxymethyl)-aza)-hexyl]-oxyj-propyl>-amide-poly-[10-(hydrazinocarbonyl )-decylamide]

If hydrazine in example 46 is replaced with an equivalent amount of 11-amino-undecanoic acid-(2-tert.-butoxycarbonyl)-hydrazide, the complex equipped with hydrazide as function is obtained

nell group.

Hydrazine content: 0.77% by weight

Analysis: C 43.30 H 7.09 N 13.36

Gadolinium complex

C 33.62 H 5.07 N 10.32 Gd 22.92

Sodium salt

C 31.63 H 4.48 N 9.74 Na 6.30 Gd 21.50

N-meglumine salt

C 37.68 H 4.61 N 9.38 Gd 14.94

Example 49

Polyethyleneimine polyacetic acid poly-<3-[4,5-di-(bis-(carboxymethyl)-amino)-2-hydroxy-cyclohexyl]-3-sulfapropyl>-amide

2.15 g (4.9 mmol) 1,2-bis-[di-(carboxymethyl)-amino]-5-(3-amino-1-thiapropyl)-4-hydroxy-cyclohexane (European patent application publication number 0154 788) is dissolved with 0.5 g of triethylamine in 50 ml of methanol and combined with a solution of 470 mg (4.75 mmol of monomeric subunits) polyethyleneimine polyacetic acid poly-methyl ester in 30 ml of methanol. After 18 hours at 60° C, the methanol is evaporated, the residue is dissolved in water, dialyzed and the retentate freeze-dried.

Yield: 2.21 g, white plates, sintering above 85°C.

Analysis: C 44.25 H 7.43 N 12.91 S 5.88

, Gadolinium complex

C 34.47 H 5.36 N 10.07 S 4.6 Gd 22.53

Sodium salt

C 33.57 H 5.05 N 9.72 Na 3.18 S 4.43 Gd 21.88

N-meglumine salt

C 36.82 H 5.05 N 9.55 S 3.63 Gd 17.77

Example 50

Polyethyleneimine polyacetic acid poly-<3-[4,5-di-(bis-(carboxymethyl)-amino)-2-hydroxy-cyclohexyl]-3-sulfapropyl>-amide-poly-[2-(maleimido)-ethylamide]

The production of the title compound takes place analogously to the prescription for example 47 by reacting polyethylenimine polyacetic acid polymethyl ester with 2-(maleimido)-ethylamine and 1,2-bis-[di(carboxymethyl)-amino)-5-(3-amino-l-thiapropyl)- 4-Hydroxycyclohexane.

Maleimide content (UV) 0.51% by weight

Analysis: C 44.43 H 7.39 N 12.99 S 5.84

Gadolinium complex

C 34.61 H 5.33 N 10.05 S 4.58 Gd 22.38

Sodium salt

C 33.49 H 5.04 N 9.79 Na 3.16 S 4.44 Gd 21.86

N-meglumine salt

C 36.96 H 5.05 N 9.55 S 3.62 Gd 17.80

Example 51

Polyethyleneimine polyacetic acid poly-<3-[4-[4,5-di-(bis-(carboxymethyl)-amino-2-hydroxy-cyclohexyl]-3-sulfapropyl>-amide polyhydrazide

The production of the title compound takes place analogously to the prescription for example 46 by reacting polyethylene-iminopolyacetic acid polymethylester with hydrazine hydrate and 1,2-bis-, [di(carboxymethyl)-amino-5-(3-amino-1-thiapropyl)-4-hydroxy- cyclohexane.

Hydrazine content (colorimetric) 0.17% by weight

Analysis: C 44.14 H 7.44 N 12.87 S 5.88

Gadolinium complex

C 34.34 H 5.36 N 10.04 S 4.6 Gd 22.55

Sodium salt

C 33.60 H 5.07 N 9.80 Na 3.17 S 4.45 Gd 21.89

N-meglumine salt

C 36.73 H 5.05 N 9.58 S 3.61 Gd 17.75

Example 52

Biotinylated gadolinium complex of poly-N-[10-carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadecanoyl]-polyethyleneimine polyhydrazide

572 mg (1 mmol) of the gadolinium complex described in example 15 is added to 20 ml of water, 220 mg (0.5 mmol) of sulfo-NHS-biotin (Pierce Chemical Company) and maintained at pH 8 - 9 by the addition of NaHCG^. After stirring for several hours at room temperature, dilute with water and dialyze until biotin can no longer be detected in the filtrate. After freeze-drying, 520 mg of colorless substance is obtained. The biotin content is determined colorimetrically (N.M. Green, Methods in Enzymology XVIII, Part A

(1970), 418 - 424): 0.39 mol%.

Example 53

a) 3,6-diaza-3,6-bis-(tert-butoxycarbonylmethyl)-4-(4-benzyl-oxycarbonylmethoxybenzyl)-suberic acid bis-(tert-butyl)-diester

93.06 g of the substance obtained in example 21a (0.15 mol) is slowly mixed with stirring with 4.48 g of NaH (80% in paraffin) (0.15 mol) in 600 ml of dry tetrahydrofuran and then added dropwise at room temperature 34.4 g bromoacetic acid benzyl ester

(0.15 mol) in 150 ml of dry tetrahydrofuran. After stirring overnight, the precipitated sodium bromide is filtered off with suction, evaporated, taken up in diethyl ether and the other inorganic components are removed by washing with water. After drying with MgSO4, the solvent is removed and the residue is purified through a silica gel column. 75.2 g (65% of theory) of a colorless oil is obtained.

b) 3,6-diaza-3,6-bis-(tert-butoxycarbonylmethyl)-4-(4-carboxymethoxybenzyl)-suberic acid-bis-(tert-butyl)-diether 9.5 g of the compound obtained in the previous reaction step (0.012 mol), is dissolved in 100 ml of dry tetrahydrofuran and hydrated in the presence of 2 g of 10% Pd/C, until there is no further hydrogen uptake. After extraction, the solvent is removed on a rotary evaporator and the substance is further dried at 0.01 torr. The viscous oil obtained weighs 8.33 g (99% of theory).

c) Poly-N6-|4-[2,3-(N,N,N',N'-tetrakis-(tert-butoxycarbonyl-methyl)-diamino)-propyl]-phenoxymethyl-carbonylj-poly-L- lysine

Dissolve 9.54 g (14 mmol) of the acid described in example 53b in DMF, add at -15° C 9.7 ml triethylamine, 1.48 ml (15.4 mmol) chloroformic acid ethyl ester and a solution of 1.16 g (7 mmol) of poly-L-lysine hydrochloride in water. The resulting suspension is stirred without further cooling, the precipitate is filtered off with suction, washed with DMF and water and then dried.

Yield: 4 g (72% of theory).

Melting point: 212° C (decomposition).

d) Poly-N<6->14-[2,3-(N,N,N',N"-tetrakis-(carboxymethyl)-diamino)-propyl]-phenoxymethylcarbonylj-poly-L-lysine. Sodium salt

3.5 g of the tetra-tertiary-butyl ester described in example 53c are suspended in 80 ml of 24% aqueous hydrogen bromide solution and stirred for 6 days at room temperature. The obtained solution is then adjusted to pH 7 with caustic soda, the residual clarity is filtered and the filtrate is ultrafiltered (lAmiconu ultrafiltration membrane YM2). After freeze-drying, 1.6 g is obtained.

Yield: 74%

Melting point: >250° C

e) Poly-N<6->(4-[2,3-(N,N,N',N'-tetrakis-(carboxymethyl)-diamino)-propyl]-phenoxymethylcarbonylJ-poly-L-lysine polyhydrazide

1.2 g (2 mmol) of the tetraacid sodium salt described in example 53d is dissolved in 60 ml of methanol/water (1:1). After adding 0.026 g (0.2 mmol) dimethylsulphate, stir for 6 hours at room temperature, then add 5 ml of 80% hydrazine hydrate solution and stir overnight. Free hydrazine is removed by ultrafiltration (pH >9) and the resulting residual solution is adjusted to a pH of approx. 4 by adding "Amberlite" IR 120 (H<+>), the ion exchanger is filtered off and the residue is freeze-dried.

Yield: 1.0 g

Hydrazide content (colorimetric): 4.8 mol%

f) Gadolinium complex of

Poly-N<6->|4-[2,3-(N,N,N',N'-tetrakis-(carboxymethyl)-diamino)-propyl]-phenoxymethylcarbonylj-poly-L-lysine polyhydrazide

1.0 g of the complex former described in example 53e is added to 100 ml of water, 253 mg of Gd2O3 and stirred for 1 hour at 80° C. The resulting solution is ultrafiltered and then freeze-dried.

Yield: 1.1 g

Gd content: 18.0% by weight

The following relaxivities were measured (the measurements of the relaxation times Tl and T2 took place in a "Minispec" p 20 (Bruker) at 0.46 Tesla (= 20 MHz), 37° C):

T-j^-relaxivity: 24.08 (L/mmol sec)

T2~relaxivity: 30.24 (L/mmol sec)

Example 54

Coupling of gadolinium complexes of poly-N<6->|4-[2,3-(N,N,N',N'-tetrakis-(carboxymethyl)-diamino)-propyl]-phenoxymethylcarbonylj-poly-L- the lysine polyhydrazide to it. monoclonal antibody 17-1A (MAK) (periodate oxidation method)

The coupling is carried out in 0.1 mol/l sodium acetate buffer pH 5, which contains 0.1 mol/l sodium chloride and is hereinafter referred to as buffer.

Dissolve 10 mg of sodium periodate-oxidized MAK in 10 ml of buffer (solution A). 1.6 g of the functionalized polymer

Gd complex from example 53g, corresponding to 0.02 mol hydrazide groups per complexing subunit (665 D), is likewise dissolved in 10 ml of buffer (solution B).

Solution A is added while shaking to solution B and the batch is incubated for 16 hours at room temperature under gentle rocking movements. The MAK concentration in the batch amounts to 0.5 mg/ml and the ratio between hydrazide and aldehyde groups is 100:1. By adding sodium cyanoborohydride (100 mol/mol MAK), the formed hydrazone is reduced.

Uncoupled Gd complex is separated from the conjugate using cation exchange chromatography in acetate buffer pH 4.5.

Example 55

Injection solution of a tumor-specific conjugate of the monoclonal antibody 17-1A and the gadolinium complex of poly-N -|4-[2,3-(N,N,N',N'-tetra-kis-(carboxymethyl) -diamino)-propyl]-phenoxymethylcarbonylj-poly-L-lysine-polyhydrazide dt

200 mg of the conjugate from Example 54 is dissolved in 10 ml of sodium bicarbonate buffer (20 mm, 130 mm NaCl). The solution is sterile-filtered and freeze-dried in a multi-medicine glass. For intranasal application, the substance is dissolved in 10 ml of sterile, double-distilled water.

Example 56

Gd complex of poly-N -[10-carboxy-3,6-bis-(carboxymethyl)-9-ethoxycarbonyl-methyl-3,6,9-triazadecanoyl]-poly-L-lysine

1.0 g of the complexing agent described in example 39a is dissolved in 100 ml of water and complexed as described in

3+

example 53f with 277 mg Gd203 = 240 mg Gd.

Yield: 1.2 g

Gd content: 19.3% by weight. ^max (H20) = 201 ^ (£=9000)

The following relaxivities were measured (the measurements of the relaxation time T1 and T2 took place in a "Minispec" p 20 (Bruker) at 0.47 Tesla (= 20 MHz), 39° C):

,T^-relaxivity: 9.61 (L/mmol sec)

T2~relaxivity: 10.56 (L/mmol sec)

Example 57

a) Poly-N 6-[10-carboxy-3,6-bis-(carbox<y>methyl)-9-ethoxycarbonylmethyl-3,6,9-triazadecanoyl]-poly-L-lysine-poly-hydrazide

2.4 g (4.2 mmol) of the ethyl ester described in example 39a is partially transferred to the hydrazide analogously to the regulation given for example 39b, and worked up at a pH value equal to or less than 9 as described there .

Yield: 2 g

Hydrazide content: 0.3 mol%, ethoxy determination: 6.3%

b) Gd complex of poly-N -[10-carboxy-3,6-bis-(carboxymethyl)-9-ethoxycarbonyl-methyl-3,6,9-triazadecanoyl]-poly-L-lysine polyhydrazide

1.9 g of the complexing agent described in example a is complexed analogously to example 53f with 525 mg O6.20^ = 455 mg Gd<3+>.

Yield: 2.3 g, Gd content: 17.0% by weight, melting point above 250° C.

Example 58

a) Sebacic acid mono(N<1->t-butoxycarbonyl-hydrazide) monomethyl ester 10.81 g (50 mmol) sebacic acid mono-methyl ester is dissolved

in tetrahydrofuran and 8.32 ml (60 mmol) of triethylamine and 5.28 ml (55 mmol) of chloroformic acid ethyl ester (ethyl chloroformiate) are added one after the other at -5° C. After 15 minutes, at this temperature, 6.61 g (50 mmol) of tert-butylcarbazate in tetrahydrofuran are added dropwise and then stirred for 2 hours at room temperature. The precipitate is filtered off, the filtrate is evaporated and taken up in ethyl acetate. The organic phase is washed successively with NaHCO 3 solution, citric acid and water and dried over Na 2 SO 4 . After evaporation of the ethyl acetate, the obtained oil is chromatographed on silica gel in diisopropyl ether.

Yield: 11.2 g (68% of theory)

b) Sebacic acid-mono(N<1->t-butoxycarbonyl-hydrazide)

9.9 g (30 mmol) of the methyl ester described in

example 58a, 150 ml of NaOH solution are added and stirred

hour at room temperature. The clear, aqueous solution is washed with ether, acidified with citric acid and the resulting precipitate is taken up in ethyl acetate. After drying over Na2S04 and evaporation of the solvent, an oily residue is obtained.

Yield: 9.3 g (98% of theory).

c) Poly-N 6 -thydrazinocarbonyl-nonanoyl]-N 6-[10-carboxy-3,6-bis-(carboxymethyl)-9-ethoxycarbonylmethyl-3,6,9-triazadecanoyl]-poly-lysine

0.48 g (1.5 mmol) of sebacic acid mono(N<1->t-butoxycarbonyl-hydrazide) (Example 58b) is dissolved in tetrahydrofuran and added at -5° C one after the other 4.16 ml (30 mmol) of triethylamine and 0.15 mL (1.58 mmol) chloroformic acid ethyl ester. After 15 minutes, a solution of 2.46 g (15 mmol) poly-L-lysine hydrochloride in water is added at -20° C. and heated to room temperature. After 3 hours, tetrahydrofuran is distilled off, diluted with water and, at pH = 9, 18.15 g (45 mmol) N 3-(2,6-dioxo-morpholinoethyl)-N c-(ethoxycarbonylmethyl)-3,6-diaza- octanoic acid (Example 13a) and then adjusted to pH = 7 with dilute hydrochloric acid. The solution is filtered, the filtrate is purified from low molecular weight components using an ultrafiltration membrane ("Amicon" YM 5) and finally freeze-dried. No contamination can be detected by thin-layer chromatography.

Yield: 6.6 g

The obtained polymeric Boc hydrazide is taken up in trifluoroacetic acid without further purification, stirred for 1 hour at room temperature and then precipitated with ether, filtered off with suction and dried. The remainder is adjusted to pH 7 in water and freeze-dried.

Yield: 5.5 g

Hydrazide content: 1.5 mol%

3+

1 g of this compound complexes 200 mg of Gd

d) Gadolinium complex of poly-N g -[hydrazinocarbonyl-nonanoyl]-N g -[10-carboxy-3,6-bis(carboxymethyl)-9-ethoxycarbonylmethyl-3,6,9-triazadecanoyl]-polylysine

5 g of the complexing agent described in the example

58c, dissolve in 500 ml of water, add 1.13 g of Gd-^O^ = 980 mg of Gd<3+> and stir for 1 hour at 80° C. The solution obtained is ultrafiltered and then freeze-dried. Yield: 5.6 g; Gd content: 16.1% by weight

Xmax (H2O)=200 nm (e = 9000)

Example 59

a) Poly-N 6 -[hydrazinocarbonyl-nonanoyl]-N 6-[10-carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadecanoyl]-poly-lysine

5.5 g of the polymeric DTPA ethyl ester described in example 58c is dissolved in as little as possible 2n NaOH solution and neutralized for 2 hours at room temperature by adding dilute hydrochloric acid. The clear solution is ultrafiltered ('Amicon" YM 5) and then freeze-dried.

Yield: 5.1 g

Ethoxy determination: negative

3+

1 g of this compound complexes 200 mg of Gd

b) Gadolinium complex of poly-N g -[hydrazinocarbonyl-nonanoyl]-N -[10-carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadeca-noyl]-poly-lysine

5 g of the complexing agent described in example 59a is complexed as described in example 58d with Gd-jO^-Yield: 5.6 g

Gd content: 16.2% by weight

Xmax (H2O) = 201 nm (e = 9000)

Example 60

aj Poly-a, (3-aspartic acid-N-[ 14-carboxy-7 ,10-bis (carboxymethyl)-

13-ethoxycarbonylmethyl-5-oxo-4,7,10,13-tetraazatetradecyl]-amide

To 2.1 g (10 mmol) a, (5-poly-aspartic acid-(N-3-aminopropyl)-amide (H.N. Kovacs et al., J. Med. Chem. 10, 904-908

(1967)) aqueous caustic soda (pH 9) is added portionwise 12.1 g

(30 mmol) N 3 -(2,6-dioxomorpholinoethyl)-N 6-(ethoxycarbonylmethyl)-3,6-diaza-octanoic acid (Example 13a). By simultaneous addition of 1 n NaOH solution, the pH in the solution is kept between 9 and 9.5. Then stir for a further 15 minutes at room temperature and

neutralized with dilute hydrochloric acid. The solution is ultrafiltered (<A>micon" YM 5) and the desalted polymer is freeze-dried.

Yield: 5.5 g (86% of theory)

Ethoxy determination: 6.33%, which corresponds to an acylation of poly-aspartic acid amides of 90%

1 g of this compound complexes 258 mg of Gd<3+>.

b) Gadolinium complex of poly-cx, |3-aspartic acid-N-[ 14-carboxy-7,10-bis(carboxymethyl)-13-ethoxycarbonylmethyl-5-oxo-4,7,10,13-tetraazatetradecyl]- amide

2.96 g (5 mmol) of the complexing agent described in example 60a is added at pH 4 - 5 in water 815 mg Gd202 =

708 mg Gd<3+> and stirred for 1 hour at 80° C. The solution obtained is ultrafiltered and then freeze-dried.

Yield: 3.30 g

Gd content: 20.5% by weight

The following relaxivities were measured [the measurements of the relaxation times T1 and T2 took place in a "Minispec" p20 (Bruker) at 0.46 Tesla (= 20 MHz), 37° C].

Tl relaxivity: 12.4 (L/mmol sec)

T2 relaxivity: 12.5 (L/mmol sec)

Example 61

a) Poly-α, |3-aspartic acid-N-[ 14-carboxy-7,10,13-tris (carboxymethyl)-5-OXO-4,7,10,13-tetraazatetradecyl]-amide

5.0 g of the polymeric DTPA ethyl ester soirrérsbés "crab"-in example 60a, is dissolved in as little as possible 2 N NaOH solution and neutralized for 2 hours at room temperature by adding dilute hydrochloric acid. The clear solution is ultrafiltered ("Amicon" YM 5) and then freeze-dried.

Yield: 4.6 g

Ethoxy determination: negative

3+

1 g of this compound complexes 2 60 mg of Gd

b) Gadolinium complex of poly-α,β-aspartic acid-N-[14-carboxy-7,10,13-tris-(carboxymethyl)-5-oxo-4,7,10,13-tetraazatetra-decyl] - amide

3.0 g of the complexing agent described in Example

61a, is complexed analogously to example 60b with Gd^^.

Yield: 3.6 g

Gd content: 20.7% by weight

The following relaxivities were measured [the measurements of the relaxation times Tl and T2 took place in a "Minispec" p20 (Bruker) at 0.46 Tesla (= 20 MHz), 37° C].

Tl relaxivity: 12.9 (L/mmol sec)

T2 relaxivity: 12.7 (L/mmol sec)

Example 62

a) 3,6-bis(carboxymethyl)-9-(N,N-diethylamino-carbonylmethyl)-3,6,9-triazaundecanedioic acid

20.17 g (50 mmol) N -(2,6-dioxomorpholinoethyl)-N -

(ethoxycarbonylmethyl)-3,6-diaza-octanoic acid (Example 13a)

add in 250 ml DMF 34.7 ml (250 mmol) triethylamine and 5.22 ml (50 mmol) diethylamine at 0° C, stir overnight at room temperature, evaporate in vacuo, dissolve in 2 N NaOH solution and dissolve in 2 hours at room temperature. The solution is applied to 'Amberlite" IR 120 (H<+>) and the acidic eluate is freeze-dried.

Yield: 14.1 g (63% of theory)

Melting point: 130° C

b) Poly-N -[10-carboxy-3,6-bis(carboxymethyl)-9-(N,N-diethyl-amino-carbonylmethyl)-3,6,9-triaza-decanoyl]-poly-L-lysine

8.97 g (20 mmol) of the acid described in example 62a is suspended in 150 ml of acetic anhydride and stirred after the addition of 9.7 ml of pyridine overnight at room temperature. The solution is reprecipitated several times from ether and the slightly yellowish powdery product is added to a solution of 1.6 g (10 mmol) of poly-L-lysine hydrochloride in 200 ml of water, whereby the pH value is maintained at 9.5 by adding diluted baking soda. It is then stirred for 1 hour, neutralized with dilute hydrochloric acid, ultrafiltered (dAmicon" YM 5) and freeze-dried.

Yield: 4.8 g (88% of theory)

Melting point: above 200°C.

3+

1 g of this compound complexes 23 6 mg of Gd

c) Gadolinium complex of poly-N<6->[10-carboxy-3,6-bis(carboxymethyl)-9-(N,N-diethylamino-carbonylmethyl)-3,6,9-triaza-decanoyl]- poly-L-lysine

4.0 g of the complexing agent described in example 62b is complexed analogously to example 60b with Gd^-j.

Yield: 4.8 g

Gd content: 19.1% by weight

Tl relaxivity: 11.75 (L/mmol sec)

T2 relaxivity: 12.93 (L/mmol sec)

Example 63

a) 3,6-bis(carboxymethyl)-9-morpholinocarbonylmethyl-3,6,9-triazaundecanedioic acid

20.17 g (50 mmol) of N 3 -(2,6-dioxomorpholinomethyl)-N 6-((ethoxycarbonylmethyl)-3,6-diaza-octanoic acid (Example 13a) is added to 250 ml of DMF 34.7 ml (250 mmol ) triethylamine and 4.8 g (55 mmol) morpholine in 20 ml DMF and worked up as described in example 62a. After freeze-drying the acidic ion exchanger eluate, 16.2 g (70% of theory) of the title compound is obtained.

b) Poly-N<6->[10-carboxy-3,6-bis(carboxymethyl)-9-morpholino-carbonylmethyl-3,6,9-triazadecanoyl]-poly-L-lysine

9.25 g (20 mmol) of the acid described in example 63a is activated analogously to example 62b with acetic anhydride/pyridine and reacted with 1.6 g (10 mmol) poly-L-lysine hydrochloride and worked up as described .

Yield: 4.8 g (85% of theory)

Melting point: above 200° C

3+

1 g of this compound complexes 230 mg of Gd

c) Gd complex of poly-N -[10-carboxy-3,6-bis(carboxymethyl)-9-morpholinocarbonylmethyl-3,6,9-triazadecanoyl]-poly-L-lysine

4.0 g of the complexing agent described in example 63b is complexed analogously to example 60b with Gd2C>2.

Yield: 4.6 g

Gd content: 18.6% by weight

T. -relaxivity: 12.31 (L/mmol sec)

T^-relaxivity: 13.15(L/mmol sec)

Example 6 4

Poly-N<6->[4-(2,5,8,11-tetrakis-carboxymethyl-2,5,8,11-tetraaza-cyclododecylmethyl)-phenoxy-hydroxypropyl]-poly-L-lysine

A solution of 2.5 g of the compound obtained according to 9e in 10 ml of tetrahydrofuran is prepared at 0° C and added dropwise at 0° C to a solution of 0.3 g of poly-L-lysine in 15 ml of water . After the addition is finished, heat for a further 1 hour under reflux. After suctioning off the solvent, it is heated in 25 ml of hot formic acid and kept for 2 hours at 50° C. The mixture is then poured into 300 ml of water, dialyzed and the retentate subjected to freeze-drying. 1.1 g of the title compound is obtained as a white powder.

Analysis: Found: C 51.88 H 6.35 N 11.77

The gadolinium complex is obtained as described in Example 38b.

Analysis: Found: C 41.33 H 4.72 N 8.91 Gd 18.88

Sodium salt

Analysis: Found: C 39.75 H 4.61 N 8.47 Gd 17.91

N-meglumine salt

Analysis: Found: C 42.55 H 4.52 N 8.53 Gd 12.97

Example 65

a) 2-(4-benzyloxycarbonylmethoxybenzyl)-1,4,7,10-tetrakis-(tert-butoxy-carbonyl-methyl)-1,4,7,10-tetraazacyclododecane

5.0 g of the compound obtained during 9d, 200 mg of sodium hydride (80% in paraffin) in 35 ml of dry tetrahydrofuran are added slowly while stirring, then 1.45 g of bromoacetic acid benzyl ester in 50 ml of dry tetrahydrofuran are added dropwise at room temperature. After stirring overnight, the precipitated sodium bromide is filtered off with suction, evaporated, taken up in diethyl ether and the remaining organic components are removed by washing with water. After drying over magnesium sulfate, it is purified through a silica gel column. After evaporation to dryness, 4.5 g of a colorless oil is obtained.

b) 2-(4-carboxymethoxybenzyl)-1,4,7,10-tetrakis-(tert.-butoxy-carbonyl 1-methyl)-1,4,7,10-tetraazacyclododecane 3.8 g of the compound obtained according to a), is dissolved in 70 ml of tetrahydrofuran and hydrogenated in the presence of 1.4 g of 10% palladium charcoal, until no more hydrogen absorption takes place. After filtration, the solvent is evaporated and the substance is dried at 0.01 torr. 1.7 g of a colorless oil is obtained. (Yield: 51% of theory) .

c) Poly-N<6->[4-(2,5,8,11-tetrakis-carboxymethyl-2,5,8,11-tetra-azacyclododecylmethyl)-phenoxyacetyl]-poly-L-lysine

At 0° C, a solution of 12.8 g of the compound obtained according to b), 2.35 g of chloroformic acid isobutyl ester and 1 g of triethylamine in 100 ml of tetrahydrofuran is prepared and this solution is added dropwise at 0° C to a solution of 1, 6 g of poly-L-lysine and 1.4 g of potassium hydroxide in 80 ml of water. After the addition is finished, the precipitate is decanted and it is treated for 2 hours at 50° C with 35 ml of hot formic acid. It is then poured into water, dialyzed and the retentate subjected to freeze-drying. 4.1 g of a white powder is obtained.

Analysis: Found: C 59.21 H 3.74 N 13.82

The gadolinium complex is obtained as described in Example 38b.

Analysis: Found: C 49.76 H 2.23 N 11.56 Gd 21.02

Sodium salt

Analysis: Found: C 47.54 H 2.11 N 11.2o Gd 19.89

N-meglumine salt

Analysis: Found: C 50.88 H 2.03 N 11.47 Gd 14.32

Example of NMR diagnostics in vivo

0.1 mmol Gd/kg was in the form of the Gd complex of poly-N-[10-carboxy-3,6,9-tris-(carboxymethyl)-3,6,9-triazadecanoyl]-poly-L-lysine -the polyhydrazide (example 39c) applied i.v. in a nude mouse (Balb/c nu/nu, female mouse, 25 g) with a subcutaneous HT29 colon carcinoma.

The substance was dissolved in 300 µl of 0.9% NaCl. The animal was examined in a nuclear spin tomograph from the company General Electric with a 2 Tesla magnet.

Recordings were made before and after application of the contrast agent with a Spin-Echo sequence (TR = 400 msec,

TC„ i = 30 msec) in the area of the liver and the tumor.

Image 1 shows the recording (transverse section) in the liver area. Picture 2 shows a transverse section through the tumor and the kidney.

Claims (11)

1. Polymer complex consisting of at least one ion of an element of the order numbers 21-29, 42, 44 or 57-83 and a carboxylic acid group-containing complex-forming polymer, characterized in that the complex-forming structure has the general formula I where A and A' each mean or with r in the meaning of the digits 0 or 1, s means the whole numbers from 7 to 20,000, t means the whole numbers from 0 to 20,000, U means a direct bond, the group V, Slf S2, S3 and S4 each mean a direct bond or one linear, branched, saturated or unsaturated C-L-C^ alkylene group which optionally contains imino, phenylene, phenylenoxy, phenyleneimino, amide, hydrazide, ester group(s), oxygen, sulfur and/or nitrogen atom(s) ) and is optionally substituted with hydroxy, mercapto, imino, epoxy, oxo, thioxo and/or amino group(s), K means a complexing agent of the general formulas IA, IB or IC: whereby n and m each stand for the digits 0, 1, 2, 3 or 4, whereby n and m together will not be more than 4, k stands for the digits 1, 2, 3, 4 or 5, 1 stands for the digits 0, 1, 2, 3, 4 or 5, q stands for the digits 0, 1 or 2, X independently each represents the residues -C00H or V where V means the residue V which exhibits a functional group at the end or a bio- or macromolecule which is bound via this functional group, whereby, if the molecule contains V, at least 0.1% of the substituent X stands for V, B, D and E, as the same or different, each stand for the group with R<2> in the sense of hydrogen or a linear, branched, saturated or unsaturated C^-C^ alkyl group which optionally contains oxygen and/or nitrogen atom(s V) and is optionally substituted with a hydroxy and/or amino group ( r), u in the sense of the digits 0, 1, 2, 3, 4 or 5, v in the sense of the digits 0 or 1, whereby B, D and E each contain a minimum of 2 and a maximum of 5 carbon atoms, Z stands for the group or the residue X, R<1> stands for the direct bond or a hydrogen atom, with the proviso that Z only stands for the group when R<1 >also means a hydrogen atom, and that Z only stands for the residue X, when R<1> also means a direct bond, W means a hydrogen atom, biotin, avidin, avidin-biotin-an ten substance, avidin-biotin-antibody fragments, the group Uw-Vw-K„, whereby Uw, Vw and K„ each have one of the meanings mentioned for U, V and K, V or the group whereby free COOH groups which are not required for complex formation of the metal ions of the elements with the specified ordinal numbers, if desired are present as a salt of an inorganic and/or organic base, an amino acid or an amino acid amide, or as an ester and/or amide. 2. Polymer complex according to claim 1, characterized in that they as polymer units contain complex-forming structures with the general formula Ia where K, W, s and t have the meaning specified in claim 1. 3. Polymer complex according to claim 1, characterized in that the functional group contained in V is the group -NH2; -NHR; -NHNH 2 ; -NRNH 2 ; -SH, -OH, -COCH3; -CH=CH-CO 2 R; whereby R and R' are the same or different and each represents a hydrogen atom, a saturated or unsaturated C1-C20 alkyl radical which is optionally substituted with a phenyl group, or a phenyl group. 4. Polymer complex according to claim 1, characterized in that the bio- or macromolecule contained in V is an antibody or antibody fragment. 5. Polymer complex according to claim 1, characterized in that the biomolecule contained in W is an avidin-biotin antibody or an avidin-biotin antibody fragment. 6. Polymer complex according to claim 1, characterized in that V stands for a -CH2-O-C6H4-CH2-; -CH2-CH(OH)-CH2-0-C6H4-CH2-; -C( =NH ) -O-C 6 H 4 -CH 2 -; - (CH2)4-NH-CO-CH2-0-C6H4-CH2-; - (CH2)4-NH-CH2-CH(OH)-CH2-0-C6H4-CH2-; -(C<H>2)3-O-C6H4-CH2-; -CH 2 -CO-NH-(CH 2 ) 3 -O-CH 2 -; -CH2-CO-NH-NH-; -CH 2 -CONH-(CH 2 ) 2 -; -CH2-CO-NH- ( CH2 )10- ; -CH 2 -CONH-(CH 2 ) 2 -S-; - (CH2)4-NH-CO-(CH2)8-; -CH2-CO-NH- (CH2 )3-NH- ; - (CH2 ) 3 - NH - gr up. 7. Process for producing polymer complexes according to claims 1 to 6, characterized in that polymers which are equipped with amino-, imino- and/or , where Fl stands for a leaving group, by alkylation, acylation, amidation and/or hydrazineation are converted into compounds which, as polymer units, exhibit structures with the general formula I' where A, A', U, V, Sx, S2, S3, S4, s and t have the above-mentioned meaning and K' is complex-forming with the general formulas I'A, I'B and I'C which are identical with the formulas given for IA, IB and IC, but in place of the substituents X and Z each carries X' and Z', whereby X' independently represents -COOH and Z' represents the group or the residue X', with the proviso that, if desired, part of the COOH groups is present as an ester and/or amide, W means a hydrogen atom, the group Uw-Vw-K'w whereby UM and Vw have the above meaning and K' w has the meaning mentioned for K', V'' or the group , whereby V' stands for the residue V which ultimately exhibits a functional group, these are then reacted in a manner known per se if desired with at least one metal oxide or metal salt of an element with the ordinal numbers 21-29, 42, 44 or 57-83 and if desired K' and/or W are transferred to K and W by conversion of at least one of the -C02H groups contained in K' or W to the desired alkylene group which exhibits a functional group at the end and possibly subsequent connection with a macro or biomolecule and/or with binding to the biotin or avidin residue, whereby the stated reaction steps (with the exception of the macro- or biomolecule linking, which can only take place after the generation of the functional group) can be carried out in a freely chosen order, and then possibly full or partial replacement of acidic hydrogen atoms still present in the polymers thus obtained complexes with cations of inorganic and/or organic bases, amino acids or amino acid amides, respectively the corresponding acid groups can be completely or partially transferred to esters or amides. 8. Process for the production of polymer complexes according to claim 7, characterized in that when the macro- or biomolecules are connected to the functionalized polymer complex or the ligand, as well as in the case of connection to the ligand, the subsequent complexation with the desired metal ions is carried out in a stationary phase. 9. Use of at least one polymer complex according to claim 1 for the production of agents for NMR, X-ray or ultrasound diagnostics. 10. Use of at least one polymer complex according to claim 7 as the hapten for the production of antibodies. 11. Diagnostic means, characterized in that it contains at least one polymer complex according to claim 1, possibly with the usual pharmaceutical additives.