IE60677B1 - Macrocyclic compounds - Google Patents

Abstract

Compounds of the general formula I <IMAGE> in which Y is a nitrogen or phosphorus atom, A<1> and A<2> are identical or different and are each a straight-chain or branched alkylene group having 2 to 6 carbon atoms, U<1>, U<2>, U<3>, U<4> are identical or different and are each a direct bond or a straight-chain or branched alkylene group having 1 to 6 carbon atoms, D<1>, D<2>, D<3>, D<4> are identical or different and are each an oxygen or sulphur atom, an alkylene group having 1 to 6 carbon atoms or a group N-R<7> where R<7> has the meaning of a hydrogen atom, a straight-chain or branched alkylene chain having 1 to 4 carbon atoms, which carries a COOR<1> group at the end, in which R<1> represents a hydrogen atom or a metal ion equivalent, D<5> has the meaning given for D<1>, D<2>, D<3> and D<4> and the group <IMAGE> where R<5> has the meaning of a hydrogen atom or of a straight-chain or branched, saturated or unsaturated C1-C20-alkylene group which may contain imino, phenyleneoxy, phenyleneimino, amide, ester groups, oxygen, sulphur and/or nitrogen atoms and may be substituted by hydroxyl, mercapto, imino and/or amino groups and has either a functional group or bound via this group a macromolecule B at the end, s and t are integers from 0 to 5, R<2> is hydrogen, a linear or branched, saturated or unsaturated, alkyl, acyl or acylalkyl group of 1 to 16 carbon atoms which is unsubstituted or substitute by one or more hydroxyl or lower alkoxy groups, or is -CH2-X-V where X has the meaning of carbonyl, of a straight-chain or branched alkylene group having 0 to 10 carbon atoms, which is unsubstituted or substituted by one or more hydroxyl or lower alkoxy groups, or of a straight &tilde& chain or branched alkylene group having 2 to 23 carbon atoms which is interrupted by oxygen atoms, V has the meaning <IMAGE> or -COOR<6> in which R<3> and R<4>, independently of one another, are hydrogen, a linear o branched alkyl group having 1 to 16 carbon atoms which is unsubstituted or substituted by one or more hydroxyl or lower alkoxy groups, or R<3> and R<4> together with the nitrogen atom represent a saturated 5- or 6-membered ring which may contain a further hetero atom and R<6> represents hydrogen or a saturated, unsaturated, straight-chain or branched or cyclic hydrocarbon radical having up to 16 carbon atoms or represents an aryl or aralkyl group, or R<2> or R<3> denotes a second macrocycle of the formula I' <IMAGE> which is bound via an alkylene chain (K) containing 2 to 20 carbon atoms, which, if desired carries carbonyl groups at the ends and, if desired, is interrupted by one or more oxygen atoms or R<1>-carboxymethylimino groups or substituted by one or more hydroxyl, lower alkoxy or carboxy-lower alkyl groups, which macrocycle can have a structure which is different from that of the parent structure, or R<2> denotes B or CH2-COB, with the proviso that if Rz represents B or CH2-COB, R<5> denotes a hydrogen atom, that at least two COORi groups are present in the molecule and that two hetero atoms of the macrocycle are each bound to at least two carbon atoms via an alkylene group and functional groups present in the molecule are, if desired conjugated with macromolecules and, if desired free carboxyl groups have been converted with organic or inorganic bases or amino acids and basic groups with inorganic or organic acids to the salts.

Description

The invention relates to the subject-matter characterised in the patent claims, that is to say, to macrocyclic complexing agents, complexes and complex salts, to agents containing those compounds, to their use as diagnostic agents and therapeutic agent's and also to a process for '4 the preparation of those compounds and agents. * Metal complexes were already being considered for use as contrast media for radiology at the beginning of the 1950‘s. The compounds used at that time were, however, 10 so toxic that there was no question of using them in humans. It was therefore extremely surprising that certain complex salts proved to be sufficiently tolerable to be considered for routine use in humans for diagnostic purposes. As the first representative of this class of compound, the dimeglumine salt of Gd DTPA (gadolinium(III) complex of diethylenetriaminepentaacetic acid) described in EP-A-71554 has hitherto proved to be very suitable as a contrast medium for nuclear spin tomography in clinical tests on over 1000 patients. The compound is 2Q primarily used in connection with diseases of the central nervous system.

An important reason for the good tolerance of Gd DTPA in clinical use is its high degree of efficacy in nuclear spin tomography, especially in the case of many brain tumours. Owing to its high degree of efficacy, Gd DTPA can, at 0.X mmol/kg body weight, be administered at very much lower doses than can, for example, X-ray contrast media in many X-ray examinations. Macrocyclic complexing agents and, especially, 1,4,7,10-tetraasa30 cyclododecane-N·' ,,N"3 -tetraacetic acid, which are the most closely related to the subject-matter of th© invention, are known from, FR-A-2539905, WO-A-8502352, C.A. 97 (1982), 2059502 and EP-A-124756.

Similar complexing agents are also described in the earlier publications EP-A-232751 and EP-A-238 196.

As a further representative of the complex salts, the meglumine salt of Gd DOTA (gadolinium(III) complex of 1,41, 7,10-tetraazacyclododecanetetraacetic acid) described in DE-A-34 01 052 has been found to be suitable for diagnostic purposes.

There is now, however, a desire to use chelates also at higher doses. This is the case especially for the purpose • 0 of identifying certain diseases outside the central nervous system by means of nuclear spin tomography (NMR diagnostics), but very especially when using chelates as X-ray contrast media.

Chelates can offer a number of advantages over iodated X15 ray contrast media: a) Radiation absorption in the higher energy range, and thus reduction of radiation stress on the patient and improvement of prerequisites for the energy subtraction method. 2q b) avoidance of the unforeseeable, and in some cases even life-threatening or fatal, so-called allergytype or cardiovascular side effects, known as "contrast media reactions, of present-day iodated X-ray contrast media.

The prerequisites are: -- high concentration of radiation-absorbing elements in the solution (X-ray) or strong influence on the NMR signals pharmacokinetics suitable for diagnostics, very secure binding of the metal ions in excretable complexes, even under in vivo conditions - good tolerance of tha highly concentrated, high5 dosage complex solution low allergic potential of all constituents of the contrast medium high degree of stability and long shelf-life of the chemical constituents of the contrast solution. >0 Those requirements apply to varying degrees and in different ways, but basically hold true for all applications of the mentioned complexes in in vivo diagnostics and, in some cases, in therapy.

The compounds according to the invention and the solu15 tions prepared therefrom fulfil the mentioned requirements in a surprising manner. They have a strong activity that can be adapted to the particular principles of the diagnostic or therapeutic method (X-ray, NMR, ultrasound, nuclear medicine) by choosing suitable metal 20 atoms.

The compounds according to the invention are used: 1. For NMR diagnostics in the form of their complexes with the ions of the transition metals of atomic numbers 21 to 29 and 42 and 44. ί5 2. For NMR and X-ray diagnostics in the form of their complexes with the ions of the lanthanide elements of atomic numbers 57 to 70 3.

Suitable for ultrasound diagnostics are both those compounds that are intended for use in NMR diagnostics and those intended for use in X-ray diagnostics . 4. For radiodiagnostics and radiotherapy in the form of their complexes with the radioisotopes of the elements of atomic number 27, 29, 31, 32,,, 38, 39, 43, 49, 62 64, 70 or 77.

Even without specific measures, their pharmacokinetics 10 permit an improvement in the diagnosis of a large number of diseases. The complexes are for the most part excreted again rapidly and unaltered so that, especially when relatively toxic metal ions are used as active ingredient, no harmful effects that can be attributed to the metal are observed in spite of a high dosage.

The practical application of the novel complexes and complexing agents is also facilitated by their adequate, and often even very good, chemical stability.

Another important advantage of the complexes and complex20 ing agents described is their extraordinary chemical versatility. Apart from by the choice of the central atom, the properties can be adapted by the choice of a wide range of substituents and/or salt-forming agents to the requirements of efficacy, pharmacokinetics, toler25 ance, ease of use, etc. For example, it is possible to attain a specificity of the compounds, very desirable in diagnostics and therapy, for structures in the organism, for certain biochemical substances, for metabolic processes, for conditions prevailing in tissue or body fluids, especially by coupling to biological substances or to substances having an interaction with biological systems. Such substances suitable for coupling may be of low molecular weight (for example glucose, amino acids, fatty acids, bile acids, porphyrins) or high molecular weight (polysaccharides, proteins, antibodies, etc.) or may also be structures that are foreign to the body but that are distributed in the body in a specific manner or that react with constituents of the body. The use of such substances will be all the more possible the more sensitive is the process of detection for a diagnostic agent or the more effective is, for example, a radioact ively labelled complex in therapy.

The compounds according to the invention can also be used in radiotherapy in the form of their complexes with radioisotopes, such as, for example, ‘92Ir. Furthermore, the complexing agents according to the invention are suitable as such or in the form of weak complexes with preferably body-specific ions (Ca24*, rig2’, Zn2~, pe2+/3-j·) for the treatment of heavy metal poisoning or certain thesaurismoses.

The raacrocyclic compounds according to the characterised by the general formula I for tetraazacyclododecane derivatives: invention are 1,4,7,10- (I) RS R2 wherein each R1, independently of the others, represents hydrogen or a metal ion equivalent, represents hydrogen or a methyl or ethyl group, represents a linear or branched, saturated or unsaturated alkyl group that has up to 16 carbon atoms and that is substituted by from 1 to 5 hydroxy or C^-C^-alkoxy groups, -CH2=X-V in which X represents carbonyl, a linear or branched alkylene group having from 1 to 10 carbon atoms that is optionally substituted by from to 5 hydroxy or C^-C^-alkoxy groups, or a linear or branched alkylene group that is interrupted by oxygen atoms and has from 2 to 23 carbon atoms, χ3 V represents -Ν , in which each of R3an(j , jq independently of the other, represents hydrogen, a linear or branched alkyl group that has up to 16 carbon atoms and that is optionally substituted by from l to 5 hydroxy or C^-C^-alkoxy groups, or R3 and R4 together with the nitrogen atom represent a pyrrolidine, piperidine, morpholine or piperazine ring, or r2 or R3 represents a second macrocycle of the formula I* that is linked via an alkylene chain that has from 2 to 20 carbon atoms and optionally carries carbonyl groups at its ends and is optionally interrupted by from 1 to 4 oxygen atoms or substituted by from 1 to 5 hydroxy, C^-C^-alkoxy or carboxy-Ci-C^-alkyl groups, or R2 represents B or CH2COB, B representing a macromolecule or bioraolecule that accumulates to an especially great extent in the organ or organ part to be investigated or in the tumour, and functional groups present in the molecule are, if desired, conjugated with foioraolecules, and the salts thereof with organic or inorganic bases or amino acids or with inorganic or organic acids.

Compounds of th® general formula I in which R1 represents hydrogen sre referred to as complexing agents and compounds of the general formula l in which at least two of the substituents R- represent a metal ion equivalent are referred to ss metal complexes.

Suitable alkyl substituents R2, R3 and R45 are saturated, unsaturated, linear or branched hydrocarbons having up to 16 carbon atoms, preferably saturated hydrocarbons which are substituted, optionally in the case of R3 and R* and by definition in the case of R2, by from 1 to 5 hydroxy 15 or lower alkoxy groups.

Alkoxy groups having from 1 to 4 carbon atoms are especially methoxy and ethoxy groups.

There may be mentioned as optionally substituted alkyl groups, for example, the methyl, ethyl, 2-hvdroxyethyl, 20 2-hydroxy-i-(hydroxymethyl)-ethyl, 1-(hydroxymethyl)ethyl, propyl, isopropenvl, 2- and 3-hydroxypropyl, 2,3dihydroxvpropyl,, butyl, isobutenyl, 2-, 3- and 4-hydroxybutyl, 2-, 3- and 4-hydroxy-2-methyIbuty 1, 2- and 3hydroxyisobutyl-, 2,3,4-trihydroxvbuty1 and 2-methoxv25 ethyl groups.

Preferred are unsubstituted alkyl groups having from 1 to 7 carbon atoms, such as, for example, the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl and hexyl groups. Also preferred are mono- and poly-hydroxy-sub30 stituted alkyl groups having from 2 to 7 carbon atoms and from 1 to 5, preferably from 1 to 4, hydroxy groups, such Q as, for example, 2- and 3-hydroxypropyl, 1,3-dihydroxyisopropvl, 1-(hydroxymethyl)-ethy1, bis- and tris-(hvdroxymethyl)methyl, 2 g 3-dihydroxy-l-hydroxymethy Ipropy 1, 2,3,4,5,6-pentahydroxyhexyl and preferably 2-hydroxy5 ethyl, 2-hydroxy-l-(hydroxymethyl)-ethyl, 2,3-dihydroxypropyl and 2,3,4-trihydroxybutyl.

When R3 and R4 together with the nitrogen atom represent a saturated five™ or six-membered ring optionally containing a further hetero atom,, represents pyrrolidine, piperidine, morpholine or piperazine.

The alkylene chain to which the second macrocycle P is linked optionally carries carbonyl groups at its ends, and contains from 2 to 20 carbon atoms. It may be interrupted by from l to 4 oxygen atom(s) or may be substituted by from X to 5 hydroxy, Ci-^-alkoxy or carboxy-Ci-«i-alkyl group(s).

Alkoxy and alkyl groups having from 1 to 4 carbon atoms are especially methoxy, ethoxy, methyl and ethyl groups.

Examples are: -(CHgG-CH252-.

-CH*}*-. -CH^-CH OH -CH2-0-CH2-. -(CHgk-. -5CH2-CMg-O-CH2-CH?i-a - UK2-G-CH2S3-. -CH?-CHs-SO-CH2-CH253-. -CH2-CH2-(0-CH2 -CH-CH0M OH OH C-L-COOK 4 ί I Λ OH OH When X represents an alkylene group having from 1 to 10 carbon atoms, the methylene group (1 carbon atom) is preferred.

If not all of the acidic hydrogen atoms are substituted by the central ion, one, several or all of the remaining hydrogen, atom(s) may be replaced by cations of inorganic and/or organic bases or amino acids. Suitable inorganic cations are, for example, the lithium ion, the potassium ion, the calcium ion and, especially, the sodium ion.

IQ Suitable cations of organic bases are, inter alia, those of primary, secondary or tertiary amines, such as, for example, ethanolamine, diethanolamine, morpholine, glucamine, Κ,Ν-dimethylglucamine and, especially, Nmethylglucamine. Suitable cations of amino acids are, for example, those of lysine, arginine and ornithine.

The complex compounds can also be linked to macromolecules that are known to accumulate to an especially great extent in the organ or organ part to be investigated. Such macromolecules are, for example, hormones, dextrans, polysaccharides, polychelones, hydroxyethyl starch, polyethylene glycol, desferrioxamines, bleomycins, insulin, prostaglandins, steroid hormones, amino sugars, amino acids, peptides, such as polylysine, proteins (such as, for example, immunoglobulins and monoclonal antibodies) or lipids (also in the form of liposomes). Special prominence should be given to conjugates with albumins, such as human serum albumin, antibodies, such cis, for example, monoclonal antibodies specific to tumour-associated antigens, or antirayosin.

Instead of the biomolecules it is also possible to link suitable synthetic polymers, such as polyethyleneimines. The diagnostic agents formed therefrom are suitable, for example, for use in tumour and infarction diagnostics.

- IU Suitable monoclonal antibodies for conjugation are especially those that are directed against antigens located predominantly at the cell membrane. Suitable as such are, for example, for tumour-imaging, monoclonal antibodies or their fragments (F(ab)2) that are directed, for example, against the carcinoembryonal antigen (CEA), human chorionic gonadotropin (β-hCG) or other antigens located on the tumour, such as glycoproteins. Also suitable are, inter alia, anti-mvosin, anti-insulin and 10 anti-fibrin antibodies.

Suitable for liver investigations or for tumour diagnostics are, for example, conjugates or clathrates with liposomes (which are used, for example, as unilamellar or multi-lamellar phosphatidylcholine-cholesterol vesicles).

The macrocvclic compounds of the general formula I are prepared as follows: in a manner known per se. in compounds of the general formula II wherein R5 is as defined above, R2 has the meaning given for R2, but is not to represent a macromolecule or biomolecule 8 or the group CH2-COB, and Z represents carboxy-protecting groups, the protecting groups Z are split off and the resulting acids (R1 in the general formula I represents hydrogen) are, if desired, a) reacted in a manner known per se with at least one 1 or b) IO or c) metal oxide or metal salt of an element of atomic number 21 to 29, 31, 32, 38, 39, 42 to 44, 49, 57 to 70 or 77 and then, if desired, acidic hydrogen atoms present are converted with inorganic and/or organic bases or amino acids and basic groups present are converted with inorganic or organic acids into physiologically tolerable salts, reacted in a manner known per se with at least one metal oxide or metal salt of an element of atomic number 21 to 29, 31, 32, 38, 39, 42 to 44, 49, 57 to 70 or 77 and then the resulting metal complexes are linked in a manner known per se via functional groups contained in the molecule or to R2 or to the CO group contained in R2 a macromolecule and, if desired, acidic hydrogen atoms present are converted with inorganic and/or organic bases or amino acids and basic groups present are converted with inorganic or organic acids into physiologically tolerable salts, linked in a manner known per se via the functional groups contained in fhe molecule or to R2 or to the CO group contained in R2 a macromolecule and then reacted, in a manner known per se, with at least one metal oxide or metal salt of an element of atomic number 21 to 29, 31, 32, 38, 39, 42 to 44, 49, 57 to 70 or 77 and then, if desired, acidic hydrogen atoms present are converted with inorganic and/or organic bases or amino acids and basic groups present are converted with inorganic or organic acids into physiologically tolerable salts.

Suitable carboxy-protecting groups Z are lower alkyl, aryl, and aralkyl groups, for example the methyl, ethyl, propyl, butyl, phenyl, benzyl, diphenylmethyl, triphenylI 2 methyl and bis(g-nitrophenyl jinethyl groups and also trialkylsilyl groups.

The protecting groups 2 are split off in a manner known per se„ for example by hydrolysis, alkaline saponifies5 tion of the esters, preferably with an alkali in agueousalcoholic solution at temperatures of from 0 to 50C or in the case of, for example, tert.-butyl esters, by means of trifluoroacetic acid.

The educts are prepared by cyclising two reactants? the resulting cyclic compounds are then, optionally after splitting off protecting groups, reacted with halogen alkanes, halogen esters or halogen acids in order to introduce the substituent R2.

The cyclisation is effected in accordance with methods known from the literature (for example Org. Synth. 58. 86 (1978), Macrocyclic Polvether Syntheses, Springer Verlag Berlin, Heidelberg, New York, 1982, Coord. Chem. Rev. 3, (1968), Ann. Chem. 1976. 916): one of the two reactants carries two leaving groups at the end of the chain and 2o the other carries two nucleophiles which displace those leaving groups. There may be mentioned by way of example the reaction of terminal dibromo-, diraesyloxy-, ditosyloxv- or dialkoxvcarbonyl-alkylene compounds containing a nitrogen atom with terminal diazaalkylene compounds containing an additional nitrogen atom in the alkylene chain.

Nitrogen atoms present are optionally protected, for example in the form of tosylates, and are freed, before the subsequent alkylation reaction, in accordance with processes known from the literature.

If diesters are used in the cyclisation reaction, then I 3 the resulting diketo compounds must be reduced in accordance with processes known to the person skilled in the art, for example using diborane.

The subsequent alkylation is carried out using halogen esters, halogen acids or halogen alkanes which may be substituted by one or more hydroxy or lower alkoxy groups and optionally contain oxygen atom(s) in the chain. The alkyl radical may also contain a terminal amino group.

If the alkylation is carried out with a dihalogenated alkane, compounds of the general formula II are formed having two macrocyclic rings connected via a carbon bridge.

Other processes known from the literature for the synthesis of compounds having more than one ring are, for example, reactions of an amine with a carbonyl compound (for example acid chloride, mixed anhydride, activated ester, aldehyde); of two amine-substituted rings with a dicarbonvl compound (for example oxalyl chloride, glutaric dialdehyde); of two rings each having a nucleophilic group with an alkylene compound carrying two leaving groups; in the case of terminal acetyls, oxidative coupling (Cadiot, Chodkiewicz in Viehe ’’Acetylenes", 597-647, Marcel Dekker, New York, 1969). The chain linking the rings can subsequently be modified by secondary reactions (for example hydrogenation)..

The hydrolysis, which may be necessary, of the ester groups formed during the alkylation with halogen esters is carried out in accordance with methods known to the person skilled in the art (for example using basic catalysts, such as alkali metal or alkaline earth metal carbonates or hydroxides).

In the alkylation with haloacetic acid, an intermediate product is obtained in which R2 = CH2X"COOH and which is converted into the monoamide by wav of the mixed anhydride using chloroformic acid ester or by means of dicvclohexylcarbodiimide and reaction with a primary or secondary amine of ths general formula R3 HN R4 Examples of suitable amines are: dimethylamine, diethylamine, di-n-propvlamine, diisopropylamine, di-n-butylamine, diisobutvlamine, di-sec,butylamine, N-methyl-n-propylamine, dioctylamine, morpholine, pyrrolidine, piperidine, pvrazoline, 2,3-dihydroxvpropylamine, H-methyl-2,3-dihydroxvpropylamine, 2hydroxy-l-(hydroxymethyl)-ethylamine, Ν, H-bis( 2-hydroxyethyl )amine, N-methyl-2,3,4,5,6-pentahydroxyhexylaraine, 2-hydroxvethylamine, 2-amino-l,3-propanediol, diethanolamine and ethanolamine.

The polyhvdroxyalky1amines can advantageously also be used for reaction in protected form, for example in the form of O-acyl derivatives or in the form of ketals. This applies especially when those derivatives are easier and cheaper to prepare than are the polvhydroxyalkylamines themselves» A typical example is the 2-amino-l-(2,2dimethyl-1,3-dioxolan-4-yl)-ethanol, the acetonide of 1araino-2,3,4-trihydroxvbutane, prepared in accordance with DE-A—31 50 917.

The subsequent removal of the protecting groups does not present problems and can be effected, for example, by treatment with an acidic ion exchanger in aqueousethanolic solution.

They The compounds of the general formula 1 in which R~ represents a hydrogen atom are complexing agents, can be isolated and purified, or they can, without being isolated, be converted into metal complexes of the general formula I in which at least two of the substituents R- represent a metal ion, equivalent.

The metal complexes according to the invention are prepared in the manner disclosed in DE-A-34 01 052 and EP-A-71504 by dissolving or suspending the metal oxide 10 or a metal salt (for example the nitrate, acetate, carbonate, chloride or sulphate) of the element of atomic number 21 to 29, 31, 32, 38,, 39, 42 to 44, 49, 57 to 70 or 77 in water and/or a lower alcohol (such as methanol, ethanol or isopropanol) and reacting with a solution or suspension of the equivalent amount of the complexing acid of the general formula I in which R1 represents a hydrogen atom and then, if desired, replacing acidic hydrogen atoms of acid groups present by cations of inorganic and/or organic bases or amino acids. Neutralisation is carried out in this process with the aid of inorganic bases (for example hydroxides, carbonates or hydrogen carbonates) of, fer example, sodium, potassium or lithium, and/or with the aid of organic bases, such as, inter alia, primary, secondary and 25 tertiary amines, such as, for example, ethanolarnine, morpholine, glucamine, N-methyl- and Ν,Ν-dimethylglucamine, and also basic amino acids, such as, for example, lysine, arginine and ornithine.

In order to prepare the neutral complex compounds, it is 30 possible, for example, to add to the acidic complex salts in aqueous solution or suspension an amount of the desired bases that is sufficient to reach neutralisation. The solution obtained can then be concentrated to dryness in vacuo. It is often advantageous to precipitate 6 the resulting neutral salts by the addition of watermiscible solvents, such as, for example, lower alcohols (methanol, ethanol, isopropanol and others), lower ketones (acetone and others), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoxyethane and others) in order thus to obtain crvstallisates that are readily isolated and purified. It has proved especially advantageous to add the desired base to the reaction mixture as early as the complexing stage and thereby to eliminate one process step.

If the acidic complex compounds contain several free acidic groups it is often advantageous to produce neutral mixed salts which contain both inorganic and organic cations as counter-ions.

That can be effected, for example, by reacting the complexing acid in aqueous suspension or solution with fhe oxide or salt of the element yielding the central ion and half the amount of an organic base necessary for neutralisation, isolating the complex salt formed, purifying it, if desired, and then adding the required amount of inorganic base for complete neutralisation. The sequence in which the bases are added can also be reversed.

Basic groups can be converted into pharmaceutically acceptable salts using inorganic and/or organic acids.

There are used as inorganic bases, for example, lithium, sodium and potassium hydroxide. Suitable organic bases are, inter alia. primary, secondary and tertiary amines, such as, for example, ethanolamine, morpholine, glueamine, Ν-methyl- and Ν,N-dimethyl-glucaraine, and also basic amino acids, such as, for example, lysine, arginine and ornithine.

I 7 Inorganic acids, such as, for example, hydrochloric acid, and organic acids, such as, for example, citric acid, are suitable for salt formation with basic groups.

Conjugate formation can be carried out, for example, by way of a carboxy group of the complex compound or by way of a functional group. When forming the conjugate of acids with macromolecules, several acid radicals can be bound to the macromolecule. In that case several central ions may be bound to one macromolecule.

Coupling to the desired macromolecules is likewise carried out in accordance with methods known per se. such as described, for example, in Rev. Roura. Morphol.

Embryol. Physiol., Physiologie 1981, 18, 241 and J.

Pharm. Sci. 68, 79 (1979), for example by reacting the nucleophilic group of a macromolecule, such as the amino, phenol, sulphhydryl, aldehyde or imidazole group, with an activated derivative of the complexing agent. Suitable activated derivatives are, for example, monoanhydrides, acid chlorides, acid hydrazides, mixed anhydrides (see, for example, G.E. Krejcarek and K.L. Tucker, Biochem. Biophvs. Res. Commun. 1977, 581), activated esters, nitrenes or isothiocyanates. Conversely, it is also possible to react an activated macromolecule with the complexing acid, Substituents having, for example, the structure C^H^NHCOCHj, C^H^NHCS or CgH^OCHjCO are also suitable for conjugation with proteins.

The conjugation of the complexing acid with dextrans and dextrins is likewise effected in accordance with methods known per se, for example by activating the polysaccharides with cyanogen bromide and then reacting with amino groups of the complexing acid.

When complex compounds containing radioisotopes are used, their preparation can be effected in accordance with the methods described in Radiotracers for Medical Applications5’ , Volume 1, CRC-Press, Boca Raton, Florida.

The pharmaceutical agents according to the invention are likewise prepared in a manner known per se by suspending or dissolving the complex compounds according to the invention, optionally with the addition of adjuvants customarily used in galenical pharmacy, in an aqueous medium and then optionally sterilising the suspension or solution. Suitable adjuvants are, for example, physiologically acceptable buffers (such as, for example, tromethamine), small amounts of complexing agents (such as, for example, diethylenetriaminepentaacetic acid), or, if necessary, electrolytes, such as, for example, sodium chloride, or, if necessary, anti-oxidants, such as, for example, ascorbic acid.

If suspensions or solutions of the agents according to the invention in water or physiological saline solution are desired for enteral administration or other purposes, they are mixed with one or more adjuvant(s) customarily used in galenical pharmacy (for example methylcellulose, lactose, mannitol) and/or surfactant(s), (for example lecithins, Tween^ , Myrj(Rb and/or flavourings to improve the taste (for example essential oils).

In principle it is also possible to prepare the pharmaceutical agents according to the invention without isolating the complex salts. Special care must in any case be taken to effect chelate formation in such a manner that the salts and salt solutions according to the invention are practically free of uncoraplexed toxically active metal ions.

That can be ensured, for example, by means of colour indicators, such as xylenol orange, by control titrations during the preparation process. The invention accordingly also relates to processes for the preparation of the complex compounds and their salts. Purification of the isolated complex salt can be used as a final safety measure.

The pharmaceutical agents according to the invention preferably contain from 1 μ mol to 1 mol/1 of the complex salt and are generally administered in doses of from 0.001 to 5 mmol/kg. They are intended for enteral and parenteral administration.

The agents according to the invention meet the many and various prerequisites for suitability as contrast media for nuclear spin tomography. For example, they are excellently suited, after oral or parenteral administration, for improving the information content of the image obtained by means of the nuclear spin tomograph, by increasing the signal intensity. They also exhibit the high degree of efficacy that is necessary in order to minimise the burden of foreign substances on the body, and the good tolerance that is necessary to maintain the non-invasive character of the investigations.

The good water-solubility of the agents according to the invention enables highly concentrated solutions to be produced so that the volume-load on the circulation can be kept within tolerable limits and dilution by body fluids can be compensated for. In addition, the agents according to the invention not only have a high degree of stability in vitro but they also have a surprisingly high degree of stability in vivo, so that a release or an exchange of the ions that are not covalently bonded in the complexes, and which are toxic per se, takes place only extremely slowly during the time in which the novel contrast media are completely excreted again.

Generally, the agents according to the invention are, for use as MMR diagnostic agents, administered in doses of from 0.001 to 5 mmol/kg, preferably from 0.005 to 0.5 mmol/kg. Details of usage are discussed, for example, in H.J. Weinmann et al., Am. J. of Roentgenology 142, 619 (1984).

Especially low doses (less than 1 mg/kg) of organspecific NMR diagnostic agents can be used, for example, for the detection of tumours and cardiac infarctions.

The complex compounds according to the invention can also advantageously be used as shift reagents.

Owing to their favourable radioactive properties and the good stability of the complex compounds contained in them, the agents according to the invention are also suitable as radiodiagnostic agents. Details of their use and dosage are described, for example, in Radiotracers for Medical Applications, CRC-Press, Boca Raton, Florida .

Another imaging method with radioisotopes is positron emission tomography which uses positron-emitting isotopes, such as, for example, 43Sc, 4^Sc, 52Fe, 55Co and 68Ga. (Heiss, W.D., Phelps, M.E., Positron Emission Tomography of Brain, Springer Verlag Berlin, Heidelberg, Mew York 1983.) The compounds according to the invention can also be used in radioimmunotherapy. This procedure differs from the corresponding diagnostics only by the amount and type of radioactive isotope used. The aim is to destroy tumour cells by high-energy short-wave radiation with as short a range as possible. The specificity of the antibody used is of crucial importance since non-specifically localised antibody conjugates lead to the destruction of healthy tissue.

The antibody or the antibody fragment of the antibodymetal complex according to the invention serves to transport the complex,, in an immunospecific manner in relation to the antigen concerned, to the target organ where the metal ion selected for its cytocidal properties can emit radiation that causes lethal damage to the cells. Suitable β-emitting ions are, for example,, i,5Sc. 7 Sc,, 48Sc, 72Ga and 73Ga. Suitable α-emitting ions having low half-life values are, for example, 2118i„ 212Si, 213Bi and 214Bi, 212Si being preferred.

In the case of in vivo administration of the therapeutic agents according to the inventiont the agents can be administered together with a suitable carrier, such as, for example, serum or physiological saline solution, and together with another protein,, such ss,- for example, human serum albumin. The dosage depends on the type of cellular disorder, the metal ion used and the type of imaging method.

The therapeutic agents according to the invention are administered parenterally, and preferably intravenously. Details of the use of radiotherapeutic agents are discussed, for example, in R.&J. Kozak et al. TI3TEC, October 1986, 262.

The agents according to the invention are outstandingly suitable as X-ray contrast media and special attention should be drawn to the fact that they do not give rise to any signs of the anaphylaxis-type reactions known for iodine-containing contrast media in biochemical-pharma22 cological studies. They are especially valuable for digital subtraction techniques owing to their favourable absorption properties in areas of relatively high tube voltages.

In general, the agents according to the invention are administered, when used as x-ray contrast media, analogously to, for example, raeglumine-diatrizoate, in doses of from 0.1 to 5 mmol/kg, preferably from 0.25 to 1 mmol/kg.

Details of the use of X-ray contrast media are discussed, for example, in Barke, Rontgenkontrastnittel (X-ray Contrast Media), G. Thieme, Leipzig (1970) and £>. Thurn, S. BGcheler - Einffihrung in die Bontgendiagnostik" (Introduction to X-Ray Diagnostics), G. Thieme, Stuttgart, New York (1977),, Because their acoustic Impedance is greater than that of body fluids and tissues? the agents according to the invention are also suitable as contrast media for ultrasound diagnostics? especially in the fora of suspensions. They are generally administered in doses of from 0-1 to 5 uaol/kg, preferably from 0.25 to l mmol/kg.

Details of the use of ultrasound diagnostic agents are described? for exaaple, in T.B. Tyler et al.. Ultrasonic Imaging 3.323 (1981), J.I. Haft, •’Clinical Echocardiography", Futura, Mount Kisco, New York 1978 and G. Stefan Bchokardiographie" G. Thieme Stuttgart/New York 1981.

In summary, it has been possible to synthesise novel complexing agents, metal complexes and metal complex salts that open up new possibilities in diagnostic and therapeutic medicine. This development would appear to be - 23 desirable above all in connection with the development of new types of imaging processes in medical diagnostics.

The following Examples serve to illustrate the subjectmatter of the invention in more detail.

In the following, the synthesis of starting material, starting with a cyclisation reaction, is described by way of examples a) 1-benzyl-4,7,10-tris (p-tolvlsulfonyl)-1.4,7.10-tetraazacvclododecane 145 g of Μ,Ν-bis[2,2·-(p-tolylsulphonyloxv))ethanebenzylamine, dissolved in 900 ml of dimethylformamide, are added dropwise, with stirring, at 100*C over a period of 3 hours to a solution of 164,5 g of the N,M = disodiura salt of Ν,Ν'',N'-tris(p-tolylsulphonyl)diethylenetriamine in 2.16 litres of dimethylformamide. 1 litre of water is then added dropwise, with stirring, at 80"c and the whole is stirred for a further 18 hours at room temperature, then cooled to 0‘C and the precipitate is filtered off with suction, washed with a small amount of ice-cold ethanol and dried at 15 torr and 60C. 175 g of the title compound are obtained.

Alternative Route 1 An analogous method for the preparation of tetraazacvclododecane derivatives can be found in M. Hediger and T.A. Kaden, Helv. Chim. Acta 66 f 861 (1983). .94 g of the N,N"disodium salt of M,N‘’,HfS-tris(Btolylsulphonyl Jdiethylenetriamine and 2 8.12 g of N-bis(2methanesulphonyloxvethyl)triphenylmethvlamine are stirred with 530 ml of dimethylformamide for 20 hours at 80-85"C, then cooled and stirred into a solution of 30 g of potassium carbonate in 5 litres of ice-water. The precipitate is filtered off with suction and the filter cake is washed with 0.5 litre of water and dried at 20C in vacuo at 150 torr. For purification, the product is dissolved in 230 ml of chloroform and 5 ml of triethylamine. The solution is filtered, concentrated .in vacuo to 200 b1; and 250 ml of ethyl acetate are added to the solution at boiling point. Th® whole is allowed to cool overnight and the precipitated crystals are filtered off with suction. 22.18 g of 1,4,7-tris(p-tolylsulphonyl)-10triphenylraethyl-i,4,7,10-tetraazacyclododecane are obtained, m.p.: 185-188’C (decomposition).

In order to split off the trityl protecting group, 31.4 g of the tricosyltrity1 derivative obtained in this manner are stirred in a mixture of 100 ml of glacial acetic acid, 75 ml of water and 300 ml of dioxane for 1 hour at 80*C. The whole is then extensively concentrated in vacuo at 60"C and diluted with 300 ml of ice-water, and 40 ral of 11N sodium hydroxide solution (pH above 12) are added. The mixture is shaken with 300 ral of chloroform, the phases are separated, the aqueous phase is extracted twice with 100 ral of chloroform each time and the combined chloroform phases are dried over sodium sulphate and concentrated by evaporation in vacuo. The foamy residue is treated with 300 ml of diethyl ether, whereupon crystallisation occurs. The crystals are filtered off with suction and dried in vacuo at SO C and 150 torr to yield 21 g of 1,4,7-tris(p-tolylsulphonyl)-1,4,7,10tetraazacyclododecane , m.p.: 202"203C.

The tritosyl compound so obtained (21 g) is dissolved in 200 ml of dimethylformamide, and 13.71 g of anhydrous potassium carbonate, 4.95 g of sodium iodide and 7.92 c of benzyl bromide are added in succession to the solution and the whole is stirred for 5 hours at 100 C, The mixture is then cooled to 20'C, stirred into 4 litres of ice-water and filtered with suction and the residue is dissolved In 2 litres of dichloromethane. The solution is extracted with 100 ml of water, dried over sodium sulphate and concentrated by evaporation in y.aC-UQ. The residue Is dissolved at boiling point in 500 al of acetonitrile and is left to crystallise out overnight.

The crystals are filtered off with suction end dried at 50’C and 150 torr to yield 16.20 g of l-benzyl-4,7,10tr is (p-toly Isulphony 1 )-1,4,7,10-tetraazacyclododecane, m.p.: 2!7-2I9''C, b) N-benzvl-1,4 ,7,10-tatraazacvclododecane 150 g of l-bensyl-4,7,10-tris(p-tolylsulphonyl)-1,4,7,15 10-tetraazacyclododecane are heated for 16 hours at 50c with 900 ral of HBr/acetic acid (40 % strength) and 125 g of phenol. After cooling to 20C, the whole is diluted with 1 litre of ether, cooled to -5C and the precipitated crystals are filtered off with suction. In order to isolate the free base, the product is dissolved in 500 ml of 4N sodium hydroxide solution, saturated with potassium carbonate and extracted several times with chloroform, dried over magnesium sulphate and concentrated by evaporation in vacuo. 39 g of fhe title compound are 25 obtained in the form of a pale yellow viscous oil. A sample was characterised as the trihydrochloride: melting point 210'C (with decomposition).

Alternative Route 2. ml of triethylamine are added at -20 C to a solution of 11.2 g of 1,4,7,10-tetraazacyclododecane in 900 ml of tetrahydrofuran and a solution of 16.2 ml off benzoyl chloride in 28© ml of tetrahydrofuran is added dropwise with stirring over 3 hours, the room temperature rising to slightly above -10’C. The whole is then stirred for 16 hours at 0-10*C, the precipitate is filtered off and the solution is concentrated by evaporation jjQ vacuo. The residue is chromatographed on 1 kg of silica gel and eluted with dioxane/water/anmonia solution (8:1:1). The fractions that are uniform according to thin layer chromatography are combined, concentrated by evaporation, dissolved in dichloromethane and filtered to remove a small degree of turbidity and the solution is concentrated by evaporation to yield 19.60 g of 1,4,,7-tribenzoyl-1,4 /7,, 10-tetraazacyclododecane f m.p.: 120—125*C. 11.5 g off the resulting tribenzoate are stirred in 150 ml of dimethylformamide with 8.3 g of anhydrous potassium carbonate, 3.0 g of sodium iodide and 7.2 ml of benzyl bromide for 18 hours at l00"C. The whole is filtered, the solution is concentrated by evaporation in vacuo. the residue is stirred twice with 50 ml of hexane each time and decanted. The hexane phases are discarded. For purification, the product is dissolved in dichloromethane and chromatographed with dichlorornethane/methanol (37:3) on 0.5 kg of silica gel. 10.2 g of 1,4,7-tribenzoyl-10-benzyl-i,4,7,10-tetraazacyclododecane are obtained, m.p.: 105-109*0.

In order to split off the benzoyl groups, the product (2.87 g) is dissolved in 290 ml of tetrahydrofuran. 11.2 g of potassium cert.-butoxide are added and the whole is heated under reflux for 4 8 hours. It is then filtered and concentrated by evaporation in vacuo and 100 ml off water are added to the residue while cooling with ice and the whole is extracted three times with 50 ml of dichloromethane each time. The combined dichlor2 7 omethane phases are shaken with 10 ml of water, dried over sodium sulphate and concentrated by evaporation in vacuo. The residue,, which is at first oily, crystallises very slowly and is triturated with 20 ml of hexane. After filtering off with suction and drying, 1.15 g ©ff Nbenzyl-1 ,7,10-tefcraazacyclododecane are obtained, m.p.: 75-7®"C. c) 1-benzyl-4,7, 10-tris f ethoxycarbonyImethy1)-1^ 7 ,JQtetraazacyclododecane 20G g of triethylamine and., over 2 hours,, 260 g off bromoacetic acid ethyl ester are added in succession af 0"’C to a solution of 131.8 g of M-benzyl-1,4,7,10tetraazacyclododecane in 1.5 litres of dichloromethane. The whole is stirred for a further IS hours at room temperature,, is shaken with 5 % sodium carbonate solution and brine, dried over magnesium sulphate and concentrated by evaporation in vacuo. The residue is dissolved in 200 ml of chloroform and filtered over 2 kg of silica gel,, elution being effected with 1 litre of chloroform/methanol (955). 210 g of the title compound are obtained in the form of a viscous oil. d) KJt , N88-tr is (ethoxycarbony lmethvl)-1,4,7, IQ-tetraazacyclododecane 100 g of l-benzyi-!' y7,,i0-tris(ethoxycarbonylmethyi)2,4,7,10-tetraazacyclododecane are dissolved in 0.5 litre of acetic acid and 0.5 litre of ethyl acetate, and 5 g of palladium-on-carbon (10 are added and the whole is shaken for 5 hours under hydrogen. The whole is filtered off from the catalyst and concentrated in vacuo. The residue is dissolved in 1 litre off chloroform, shaken with 100 ml off saturated soda solution and 100 ml of brine, dried and concentrated by evaporatioife in vacuo.

The residue is purified by bulb tube distillation at 10~J torr and 120’C. H(,H/,H"-tris(ethoxycarbonylmethyl)~· X 7,. IQ-tetraazacyclododecane is obtained in the form of a pale yellow viscous oil.

Yield: 65 g IS; (filsa): 3400, 2935, 2678, 1738/cse.

ExamolS-JL Gadolinium III) complex of N-C2()3-diiiydroxy-N-niethylpropylcarfoaraoylmethylJ-l, 4,7,10-tetraazacyclododecaneM' , ,H"?l-triacetic acid g of N-( 2 g 3-dihydroxy-N-raethylpropylcarbamoylraethyl ]N* ,N’9 ,Ν’1' -’-trisCethoxycaroonylmethyl )™1 „ 4,7,10-tetraazscyclododecane are dissolved in 0.5 litre ot ethanol, and 96 ml of 3N sodium hydroxide solution are added to the solution and the whole is stirred for 3 hours at 20’C and then concentrated in vacuo. 300 ml of water are added and the pH is adjusted to 6 with 2N hydrochloric acid. 31.94 g of gadolinium acetate are added to the solution and the whole is stirred for 18 hours at 50Cz then 2o passed over an anion exchanger Amberlite IRA 410 and then the aqueous eluate is passed over a cation exchanger Amberlite IRC 50.

The eluate is concentrated by evaporation in vacuo and dried. 47.14 g (73 % of the theoretical yield) of the title compound are obtained in the form of a colourless powder.

Analysis: C2o K34 Gd N5 O© (545.77) C 37.20 H 5.31 Gd 24.35 H 10.85 (calc.) 37.52 5-19 24.09 10.87 (found) The starting material for th® preparation of th© title - 29 compound according to Example 2 is obtained in the following manner: a) M' ,N"-tris(ethoxycarbonylmethyl)-ls4,7,10-fcetraazacyclododecane-X4’ w-aeetie acid g of $,N',N*-tris(ethoxycarbonylmethyl)-1,4,7,10tetraazacyclododecane are dissolved in 300 ssl of dichloromethane. 10.40 g of triethylamine are added and then a solution of 4.40 g of chloroacetic acid in 100 ml of diehloromethane is added dropwise at 0C and the whole is stirred for 20 hours at room temperature. For workingup, the mixture is partitioned between diehloromethane and phosphate buffer pS 6, dried over magnesium sulphate and concentrated by evaporation in vacuo. 23 g of the desired compound ar® obtained in the fora of a viscous oil. to) N-( 2,3-dihydroxy-N-methylpropylcarbamoylmethyl )N ' „ bP ,N 3-tris(ethoxycarbonylmethyl )~1,4,7,10-tetraazacyclododecane ,5 g di triethylamine and then a solution of 14 g of chloroformic acid isobutyl ester are added dropwise at 0"C to a solution of 48.85 g (100 mmol) of Ν,Ν’ ,.Ν7tris (ethoxycarbonylmethyl) -1., 4 ,7, IQ-tetraazacycloclodecane-N1 ’’-acetic acid in 500 ml of diehloromethane.

The whole is stirred for 1 hour at 0C and then a solution of 10.52 g of N-methylamino-2,3-propanediol in 10-0 stul of chloroform is added dropwise thereto and stirred for 2 hours at roo». temperature. After shaking with sodium hydrogen carbonate solution and brine and drying over magnesium sulphate, the product is concentrated by evaporation in vacuo. For purification, th© chloroform solution of the residue is filtered over 500 g of silica gel to yield 55 g of the title compound.

- JO £xample_2 Gadolinium(III) complex of N"(N-ethylcarbamoylmethyl]" 1,4,7, 10-tetraazacyclododecane-N*· -triacetic acid g of ^-(N-ethylcarbaieoyli®ethyl)-'S*,HH?JN8’'f-’trisiethoxy" carbonylmethyl)*-l,4,?,10-tetraazacyclododecane are . , dissolved in 250 ®1 of ethanol. 47 al of 3N sodium hydroxide solution are added to the solution and the whole is stirred for 3 hours at 20*C and concentrated in vacuo. 150 wl of water are added and the pH is adjusted to 6 with 2N hydrochloric acid. 15.55 g of gadolinium acetate are added and the whole is stirred for 5 hours ait 60 Ό. The solution is then purified by means of ion exchangers as described in the preceding Examples. .39 g of the title compound are obtained in the form of a colourless powder.

Analysis: Ciq H3Q Gd Q'j (585.72) C 36.91 H 5.16 Gd 26.85 N 5.16 (calc.) 36.98 5.28 26.58 5.07 (found) Preparation of the starting materialϊ g (51„2 mmol) of N,N’,Nw-tris(ethoxycarbonylmethyl) 1,4,7 e XO-tetraazacyclododecane-N‘’-acetic acid (see Example 2a) are dissolved in 200 ml of dichioromethane, and then 5.40 g of triethylamine followed by 7 g of chloroformic acid isobutyl ester are added at O C, The whole is stirred for 1 hour at 0C and then a solution of 2.31 g of ethylamine in 20 ml of dichioromethane is added dropwise. The mixture is stirred for 2 hours at room temperature and, after shaking with saturated sodium hydrogen carbonate solution and brine and drying over magnesium sulphate,, th© product is evaporated to dryness in vacuo For purification, the dichioromethane solution I of the residue is filtered over 200 g of silica gel to yield 24 g of N-(H-ethylcarbemoylmethyl)-N/i,N'ssN‘ja'’™ tris(ethoxycarbonyImethyl )-1,4,7, lO-tetraasacyclododecane.

Gadolinium (ill) complex of N-<2,3-dihydroxy-l-propyl)1,4,7, 10-tetraazacyclododecane-N,Η*,"*-triacetic acid 4.30 g of N,N*,N-tris(ethoxycarbonylmethyl)-l,4,7,10tetraazacyclododecane are dissolved in 100 ml of di10 chloromethane, and 4.20 g of triethylamine and 2.21 g of 3-chloro-l,2-propanediol are added. The «hole is stirred for 15 hours at room temperature and,, after shaking with water, the product is concentrated by evaporation in vacuo. The residue is stirred for 6 hours with 100 ml of IN sodium hydroxide solution, the pH is adjusted to 5 with 2N hydrochloric acid and the whole is stirred for 16 hours with 3.34 g of gadolinium acetate at 50‘C The resulting solution is subjected to ion exchanger purification to yield 3..62 g of the title compound in the form of ® colourless powder.

Analysis: C7 H2e Gd N4 O3 (574.69) C 35.53 H 5.09 Gd 27.36 N 9.75 (calc.) .68 5.19 27.03 9.68 (found) Example 4 Gadolinium^ III) complex of N-(2-arainoethyl)-1,4,7,10tetraazacyclododecane-Nt M' f N8! '-triacetic acid 2.10 g of triethylamine and 1.22 g of N-(2-chloroethyl)acetamide are added to a solution of 4.30 g of Ν,Η',Νtris (ethoxycarbonylaethy 1 )-1 „ 4,7,, 10-tetraazacyclododecane in 100 ml of dichloromethane. The whole is stirred for 18 hours at room temperature, shaken with water and concentrated by evaporation in vacuo. The residue is stirred for 8 hours with 50 ml of IM sodium hydroxide solution at 6©*C, the pH is adjusted to 6 with 2N hydrochloric acid and the whole is stirred for 16 hours with 3.34 g of gadolinium acetate at SO'C. The resulting solution is subjected to ion exchanger purification to yield 3.21 g of the title compound in the form of a colourless powder.

Analysis: C16 M2g Gd Ms Oe (543.68) C 35.35 H 5.19 Gd 28.92 N 12,88 (calc.) .17 5.45 28.68 12.81 (found) Example 5 Sis-gadolinium(III ) complex of X,1^-(1,3-propylene)1 5 bis( 1 3,7.. 10-tetraazacyclododecane-4 ., 7,10-triacetic acid) 2.02 g of 1,3-dibromopropane are added to a solution of 8.60 g of Ν, N * ,N"-tris (ethoxycarbonv lmethyl)-1 , 4,7,10fetraazacyclododecane and 4.2 g of triethylamine in 200 ml of dichloromethane and the whole is stirred for hours at room temperature. After shaking with water and brine,, the product is concentrated by evaporation in vacuo. The residue is filtered with dichloromethane over 150 g of silica gel and concentrated by evaporation. A viscous oil is obtained which is stirred with 60 ml of IM sodium hydroxide solution for 16 hours and then diluted with 100 sal of water and adjusted to pH 6 with 2N hydrochloric acid. After the addition of 6.68 g of gadolinium acetate, the whole is stirred for 16 hours at 50"C and 30 the solution is purified by means of anion and cation exchangers. 6.56 g of the title compound are obtained in th© form of a colourless powderAnalysis: C3i H^g Gd2 Ng Oj2 (1041.28) C 35.76 H 4.84 Gd 30.20 N 10.76 (calc.) .71 4.58 29.94 10.88 (found) :3xs-c;adoliniui«i( III) complex of succinyl-bis( 1,4,7,10tetraazacyclododecane-4,710-triacetic acid) 1.705 g of succinic acid dichloride dissolved in 20 ml of dichloromethane are added dropwise at oC to a solution of 4.30 g of M 8 ,He3"tris(ethoxycarfoonylraethyl)-X,4 ,7 i710-tetraazacyclododecane and 4.20 g of triethylamine in 100 ral of dichloromethane and the whole is then stirred for 1 hour at roora temperature. After shaking with sodium hydrogen carbonate solution and. brine, the product is concentrated by evaporation in vacuo. The residue is chromatographed on 100 g of silica gel with dichloromethane/ethyl acetate (0 - 30 %).

A viscous oil is obtained which is stirred for 4 hours with 40 ml of IM sodium hydroxide solution, then diluted with water (100 ml) and adjusted to pH 6 with 2M hydrochloric acid. After the addition of 3.34 g of gadolinium acetate, the whole is stirred for 16 hours at 50’C and the solution is purified by means of ion exchangers to yield 3.90 g of the title compound in the form of a white powder.

Analysis: C32 H^q Gd2 Mg Oi.4 (1083.28) C 35.48 d 4.47 Gd 29.03 M 10.34 (calc.) .31 4.18 28.81 10.57 (found) Exampig_7 M-(2-hydroxyethy1)-1,4,7,10-tetraazacyclododecaneNe ,N,N"*-triacetic acid 3.2 g of triethylamine and 1.45 g of 2-chloroethanol are added to a solution off 6.46 g of HpH'is (ethoxycarbony Imethy 1 )-1,4 „ 7,, 10-tetraazacyclododecane in 150 sal of dichloromethane. The whole is stirred for 4 hours at room temperature and, after shaking with sodium hydrogen carbonate solution and brine, the product is dried over magnesium sulphate and concentrated by evaporation in, vacuo. The residue is stirred for 16 hours with 60 ml of IN sodium hydroxide solution. The pH is adjusted to 2.5 by the addition of 5N hydrochloric acid. The resulting suspension is poured onto an ion exchanger (DOWEX 50W-X4 in the K': form) and then eluted with water followed by 0 5 H NH3 solution. The whole is concentrated in vacuo and the title compound is isolated by adding ethanol and filtering off the precipitate with suction. 4.24 g of the title compound are obtained, the purity of which is checked by pH titration and elemental analysis.

Analysis: Ci6 H30 O7 (390.44) C 49.22 H 7.75 N 14.35 (calc.) 49.48 7.83 14.09 (found) Example 8 N-(N-(2-hydroxyethy1)-carbamoylmethyl )-1,4,7,10-tetraazacyclododecane-N *, ΝΒβ, N,s *-triacetic acid .22 g of triethylamine and then 3.50 g of chloroformic acid isobutyl ester are added at 0"C to a solution of 12.50 g of N, N£’, N8’-tris (ethoxycarbony Imethy 1)-1,4,7,103 5 tetraazacyclododecane-N'-acetic acid in 250 ml of dichlorornethane. After 1 hour, 1.60 g of ethanolamine dissolved in 50 ml of dichlorornethane are added dropwise, the whole is stirred for 2 hours at room temperature and, after shaking with soda solution and brine, the product is dried over magnesium sulphate and concentrated by evaporation in vacuo. The residue is chromatographed with chloroform/acetone (10si) on 200 g of silica gel to yield 11 g of N-(2-hydroxyethyl )carbamoylmethyl-M’,Ηι,Ν85,?tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacvclododecane.

The product is stirred for 5 hours at room temperature with 100 ml of IN sodium hydroxide solution and then acidified to pH 2.5 with dilute hydrochloric acid and the suspension is purified by means of a cation exchanger (DOWEX 5OW-X4), elution being effected with water and then with 0.5H NH3 solution. The eluate is extensively concentrated and, after the addition of ethanol, the title compound crystallises out and is isolated by filtration. 7.2 g of the title compound are obtained, the purity of which is examined by titration and elemental analysis.

Analysis: C^g H33 Og (447.49) C 48.31 H 7.43 N 15.65 (calc.) 48.20 7.48 15.49 (found) Example 9.

Gadolinium complex of M-(raorpholinocarbonyImethyl)1,-4.7, lO-retraazacyclododecana-W',, Ν· , N"! -triacetic acid 55.77 g of N-(morpholinocarbonvlmethyl)-N tris (ethoxycarbonylmethyl)-1,4,710-tetraazacyclododecane are hydrolysed analogously to Example 1 with sodium 3ό hydroxide solution and complexed with gadolinium acetate 45.19 g (72 % of the theoretical yield) of the title compound are obtained in the form of a white powder.

The starting material for the preparation of the title 5 compound is obtained by proceeding as in Example 2b but using morpholine instead of N-methyIamino-2,3-propane·· diol.

Analysis: C2o H32 Gd N5 Og (627.75) C 38.27 H 5.14 Gd 25.04 N 11.16 (calc.) 38.02 5.09 24.83 11.35 (found) Example 10 Gadolinium complex of N-(morpholinocarbonylraethyl)1,4,7, lO-tetraazacyclododecane-N* »N’!, e -tris(2-methylacetic acid) 53.06 g of N-(morpholinocarbonyImethy 1)-N* ,NM,NW*-tris(1 ethoxycarbonyl-l-ethy 1 )1,4,7,xo-tetraazacyclododecane are hydrolysed analogously to Example 2 with sodium hydroxide solution and complexed with gadolinium acetate 52.20 g (78 % of the theoretical yield) of the title compound are obtained in the form of a white powder.

Analysis; C23 H3Q Gd N5 Og (669.83) C 41.24 H 5.72 Gd 23.48 N 10.46 (calc.) 41.35 5.65 23.33 10.62 (found) Example 11 Bis-gadolinium complex of X , 1 *--(2-hydroxy-l e 3-propylene) bis(1,4,7,10-tetraazacyclododecane-4 ,7,10-triacetic acid) 7 4.63 g of epichlorohydrin are added to a solution of 43.05 g of N, Hz, N'"-tris( ethoxycarbonylraethyl )-1, & , 7,10tetraazacyclododecane in 450 ml of dimethylformamide. After 1 hour, 7.5 g of sodium iodide are added and the whole is heated for 24 hours at 80C and then concentrated in vacuo. Th® residue is partitioned between water and chloroform, the chloroform phase is dried over sodium sulphate and concentrated by evaporation in vacuo. The residue is chromatographed on 1 kg of silica gel with dichlororaethana/10% acetone to yield 27.5 g of 1,1*-(2hydroxy-1,3-propyiene)-bis(l ,4,7. 10-tetraazacyclododecanetriacetic acid ethyl ester) in the form of a viscous oil. 9.17 g of the resulting ester are dissolved in 200 ml of ethanol and stirred for 20 hours at room temperature with 30 ml of 3N sodium hydroxide solution, then adjusted to pH 5 with hydrochloric acid and stirred for 16 hours at 60 C with 6.68 g of gadolinium acetate and the solution is purified by means of anion and cation exchangers to yield 10.05 g of the title compound in the form of a white powder.

Analysis: C31 H50 Gd2 Ng O13 (1057.28) C 35.22 H 4.77 Gd 29.75 N 10.60 (calc.) .03 4.89 29.49 10.41 (found) Example 12 M-methylglucamine salt of the manganesa( II) complex of N[N-(2-hydroxyethyl)-carbamoylmethyl 3-1,4,7,10-tetraazacyclododecane-N f ,Η",N"g-triacetic acid 8.95 g (20 mmol) of 2-hydroxyethyl)-carbamoylmethyl 3—1,4,7,10-tetraazacyclododecane-N *’, N, NM’ -triacetic acid are suspended in 30 ml of water and heated with 1.40 g (20 mmol) of manganese(11) oxide for 3 hours at 100"c. 3.90 g (20 mmol) of N-raethylglucamine are then added and, after heating for a further 12 hours at 100C, the solution is evaporated to dryness in vacuo. 13.8 g of the title compound are obtained in the form of a pink powder, m.p. 14O-143*C.

Analysis: C25 H^g Μη N6 0i3 (695.64) C 43.17 H 6.96 Mn 7.90 N 12.08 (calc.) 43.44 7.16 7.69 12.01 (found) Example 13 Dvsprosium(III) complex of N-(morpholinocarbonylmethyl)1,4,7,10-tetraazacyclododecane-N',Ν",Ne-triacetic acid g of H-(morpholinocarbonylmethy1)-N',N",Me-tris(ethoxycarbonyImethyl )-1,4,7,10-tetraazacyclododecane are hydrolysed analogously to Example 2 with sodium hydroxide solution and complexed with dysprosium acetate. 16.3 g of the title compound are obtained in the form of a white powder.

Analysis Cnn n32 C 37.95 37.71 Dy W5 Og (633.01) H 5.10 Dy 25.67 4.92 25.81 N 11.06 11.32 (calc.) (found) Example 14 Preparation of liposomes loaded with gadolinium-^(morpholinocarbonylmethyl)-1,4,7,10-tetraazacyclododecane-N ' , N" ,, N8’' -triacetic acid A lipid mixture of 75 mol % egg phosphatidylcholine and mol % cholesterol are prepared in the form of a dry substance in accordance with the procedure described in Proc. Natl. Acad. Sci. U.S.A. 75, 4194. 500 mg thereof are dissolved in 30 ml of diethyl ether, and 3 ml of an aqueous 0.1M solution of the gadolinium complex of M(morpholinocarbonylmethy1 )-1,4,7,10-tetraaza-Nf, Nsa, N"f triacetic acid are added dropwise thereto in an ultrasound bath. The ultrasound treatment is continued for another 10 minutes and then concentration jjj vacuo is carried out. The gel-like residue is suspended in 0.125M sodium chloride solution and repeatedly centrifuged at 0’C and 20,000 g in order to separate non-encapsulated gadolinium complex. The suspension is then subjected to freeze-drying in multivials. Administration is effected in the form of a colloidal dispersion in 0.9 % sodium chloride solution.

Example 15 Preparation of a solution of the yttrium-90 complex of the conjugate of 1,4,7,10-tetraazacyclododecane-N ,N'„N,S, Ne-tetraacetic acid with monoclonal antibodies mg of N-(3-dimethylaminopropyl)-Nz-ethylcarbodiimide hydrochloride and then 1 ml of a solution of 0.6 mg of monoclonal antibodies (with specificity againsc melanoma antigen) dissolved in 0.05 molar sodium hydrogen carbonate buffer (pH 7.8) are added to a suspension of 4 mg of 1,4,7,10-tetraazacyclododecane-N,Ν',N",N"'-tetraacetic acid in 1 ml of water. The whole is stirred for 2 hours at room temperature and dialysed against a 0.3M sodium phosphate buffer. 1 ral of an yttrium-90 solution in acetate buffer pH 6 (prepared in accordance with Int. J. Appl. Radiat. Isot., Vol 36 [1985], p. 803) is then added and the whole is incubated for 24 hours at room temperature. The solution is passed over a Sephadex G 25 column and the radioactive protein fraction is sterile-filtered and introduced into multivials. A storable dry prepare-.4 0 tion is obtained by lyophilisation.

Example 16 Indium(III) complex of M~(morpholinocarbonylmethyl)1,4,7,10-tetraazacyclododecane-Ne „ Nu' ,Mn*-triacetic acid The procedure is analogous to Example 9 and complexing is effected with radioactive mindium chloride. In order to check whether the metal ions have been completely bound in the form of a chelate, a solution of the title compound is investigated by thin-layer chromatography on silica gel plates in the system methanol/water (2:1).

Mon-chelated metal ions are recognised as a radioactive zone at the starting spot. If necessary, the chelation is completed by adding more N-(morpholinocarbonylmethyl)~ N',N",N"‘-tris(ethoxycarbonyImethyl)-1,4,7,10-tetraaza15 cyclododecane and subsequent ester cleavage.

The gadolinium-153 complex of N-(morpholinocarbony1methyl)-1,4,7,10-tetraazacyclododecane-N',NM,N'-triacetic acid is obtained in the same manner.

Claims (16)

1. Patent Claims

1. X,4,7,XO-tetraazacyclododecane derivatives of the general formula I (I) i^OQC wherein each R 1 , independently of the others, represents hydrogen or a metal ion equivalent, R 5 represents hydrogen or a methyl or ethyl group, R 2 represents a linear or branched, saturated or unsaturated alkyl group that has up to 16 carbon atoms and that is substituted by from 1 to 5 hydroxy or C-,-C,-alkoxy groups, -CH 2 ”X-V in which X represents carbonyl, a linear or branched alkylene group having from 1 to 10 carbon atoms that is optionally substituted by from 1 to 5 hydroxy or Ci~C 4 -alkoxy groups, or a linear or branched alkylene group that is interrupted by oxygen atoms and has from 2 to 23 carbon atoms, V represents -Ν , in which each of R 3 and R 4 , independently of the other, represents hydrogen, a linear or branched alkyl group that has up to 16 carbon atoms and that is optionally substituted by from l to 5 hydroxy or Ch-C^-alkoxy groups, or R 3 and R 4 together with the nitrogen atom represent a pyrrolidine, piperidine, morpholine or piperazine ring, or R 2 or R 3 represents a second macrocycle of the formula I 1 a ’ 0 0 C—\ / that is linked via an alkylene chain that has from 2 to 20 carbon atoms and optionally carries carbonyl groups at its ends and is optionally interrupted by from 1 to 4 oxygen atoms or substituted by from 1 to 5 hydroxy, CH-C^-alkoxy or carboxy-Cj-C^-alkyl groups, or R 2 represents B or CK 2 -CO3, B representing a macromolecule or bioraolecule that accumulates to an especially great extent in the organ or organ part to be investigated or in the tumour, and functional groups present in the molecule are, if desired, conjugated with biomolecules,, and the salts thereof with organic or inorganic bases or amino acids or with inorganic or organic acids,

2. Compounds according to claim 1, characterised in that each R 1 represents a hydrogen atom.

3. Compounds according to claim 1,, characterised in that at least two of the substituents R 1 are metal ion equivalents of at least one metal of atomic number 21 to 29,, 42, 4 4 or 57 to 70.

4. Compounds according to claim 1, characterised in that at least two of the substituents R^· are metal ion equivalents of at least one radionuclide of an element of atomic number 27, 29, 31 32, 38, 39, 43, 49, 64, 70 or 77.

5. N“[2, S'-dihydroxy-N-methylpropylcarfoamoylmethvl ]- 43 1,4,7,iQ-tetraazacyclododecane-N',Μ,Ν'-triacetic acid, Ν-(N-ethylcarbamoyImethyl)-1,4,7,10-tetraazacyclododecane-M* ,M IS ,H '-triacetic acid, N-(2,3-dihydroxy-l-propyl)-l,4,7, 10-tetraazacyclo5 dodecane-N'lacetic acid,, N-( 2-aminoethyl )-1,4 ,, 7, 10-tetraazacyclododecane-H 3 ,N W , N” -triacetic acid, N-(2-hydroxyethyl)-1,4,7,io-tetraazacyclododecane~ N £? ,N·’„N' e -triacetic acid, 10 N-(N-(2-hydroxyethyl)-carbamoylmethyl)-1,4,7,10-teiraazacyclododecane-N' ,Ν,Ν*’-triacetic acid, N-( morpholinocarbonyInethyl)-1,4,7,10-tetraazacyclododecane-N' ,N” ,N”'' -triacetic acid, N-(morpholinocarbonvlmethyl)-1,4,7,10-tetraazacyclo15 dodecane-N',N,N'-tris-(2-methvlacetic acid), and the complexes of those compounds with paramagnetic metals according to claim 2 or radioisotopes according to claim 4.

6. 1,1'-(1,3-propylene)-bis (1,4 e 7,10-tetraazacyclo20 dodecane-4,7,10-triacetic acid), succinyl-bis(1,4,7,10-fcetraazacyclododecane-4,7,10triacetic acid), 1,1^--( 2-hydroxy-l, 3-propylens) - bis (1,4,7, XO-tetraazacyclododecane*-4,7,10-triacetic acid), and the complexes of those compounds with paramagnetic metals according to claim 3 or radioisotopes according to claim 4.

7. Gadolinium, manganese and dysprosium complexes of the 5 complexing agents mentioned in claims 5 and 6.

8. Pharmaceutical agents containing at least one metal complex of the general formula I in which at least two of the substituents R 1 represent a metal ion equivalent, optionally in the form of liposomes and optionally with 10 the adjuvants customarily used in galenical pharmacy.

9. Use of the compounds according to claim 2 as complexing agents.

10.. Use of at least one metal complex of the general formula I in which at least two of the substituents R 1 15 represent a metal ion equivalent for the preparation of media for NMR diagnostics, X-ray diagnostics, ultrasound diagnostics or radiodiagnostics and radiotherapy.,

11. Process for the preparation of 1,4,7,10-tetraazacyclododecane derivatives according to claims Ito 3, 20 characterised in that, in a manner known per se, in compounds of the general formula XI wherein R5 is as defined above, 25 R 2 ** has the meaning given for R 2 , but is not to repres- 45 ent a macromolecule or bionolecule B or the group CH2COB, and Z represents carboxy-protecting groups, the protecting groups Z are split off and the resulting acids {R 3 · in the general formula X represents hydrogen) are, if desired, a) . reacted in a manner known per se with at least one metal oxide or metal salt of an element of atomic number 21 to 29, 31, 32, 38, 39, 42 to 44, 4$, 57 to 70 or 77 and then, if desired, acidic hydrogen atoms present are converted with inorganic and/or organic bases or amino acids and basic groups present are converted with inorganic or organic acids into physiologically tolerable salts, or b) reacted in. a manner known per se with at least one metal oxide or metal salt of an element of atomic number 21 to 29, 31, 32, 38, 39, 42 to 44, 49, 57 to 70 or 77 and then the resulting metal complexes are linked in a manner known per se via functional groups contained in the molecule or to R 2 or to the CO group contained in R 2 a nacromolecule and, if desired, acidic hydrogen atoms present are converted with inorganic and/or organic bases or amino acids and basic groups present are converted with inorganic or organic acids into physiologically tolerable salts, or c) linked in a manner known per se via the functional groups contained in the molecule or to R 2 or to the CO group contained in R 2 a macromolecule and then reacted, In a manner known per se„ with at least one metal oxide or metal salt of an element of atomic number 21 to 29, 31, 32, 38, 39,, 42 to 44, 49, 57 to 70 or 77 and then, if desired, acidic hydrogen 46 atoms present are converted with inorganic and/or organic bases or amino acids and basic groups present are converted with inorganic or organic acids into physiologically tolerable salts. 5

12. Process for the preparation of the pharmaceutical agents according to claim 8, characterised in that th® complex compound dissolved or suspended in water or physiological saline solution is made into a form suitable for enteral or parenteral administration, 10 optionally together with adjuvants customarily used in galenical pharmacy.

13. A compound substantially as hereinbefore described with reference to the Examples.

14. A pharmaceutical agent substantially as hereinbefore 15. Described with reference to the Examples.

15. A use substantially as hereinbefore described with reference to the Examples.

16. A process substantially as hereinbefore described with reference to che Examples.