FI94758B - Macrocyclic compounds, having a 3,6,9,15-tetra-azabicyclo / 9.3.1 / pentadecane structure, their use in NMR, X-ray or radio diagnostic useful composites, and such compositions - Google Patents

Abstract

6-Membered-ring-containing macrocyclic tetraaza compounds of the general formula I <IMAGE> in which ... represents a single or double bond, Q represents a nitrogen atom or the radical NH, X<1> represents a hydrogen atom, -(CH2)n-R<1>- or <IMAGE> group where n denotes the figures 1 to 5, m denotes the figures 0 to 2 and R<1> denotes a hydrogen atom or a hydroxyl group, X<2> represents X<1> or a -(CH2)n-(O)l-(CH2)k-(C6H4)q-R<2> group where k denotes the figures 0 to 4, l and q denote the figures 0 or 1 and R<2> denotes a hydrogen atom, a C1-C4-alkoxy group, a functional group or, bonded by means of this functional group, a bio- or macromolecule, A<1>, A<2>, B<1>, B<2>, C<1>, C<2>, D<1>, D<2>, E<1>, E<2>, F<1> and F<2> independently of one another each represent X<2> G represents R<2> or a second macrocycle, bonded by means of K, of the general formula II <IMAGE> where K denotes a direct bond, a bis(carbonylamino) group (-NH-CO-CO-NH-) or a C1-C14-alkylene group which optionally at the ends carries carbonyl( &rdurule& CO) or carbonylamino (-NH-CO-) groups or oxygen atoms and optionally contains one or more oxygen atom(s), hydroxymethylene (-CH-OH-), CH(X<2>)COOZ, acyl or hydroxyacyl- substituted imino groups or one or two C-C double and/or C-C triple bonds, Z represents a hydrogen atom and/or a metal ion equivalent of an element of atomic number 21-29, 31, 32, 37-39, 42-44, 49 or 57-83, with the proviso that the 12 ring substituents A<1> to F<2> represent at least 8 hydrogen atoms, that X<1> and X<2> only simultaneously represent hydrogen atoms when at least one of the ring substituents A<1> to F<2> does not represent a hydrogen atom and that the macrocycle of the general formula I no longer contains a bio- or macromolecule and that, if desired, the radical of the CO2H groups is present as ester or amide, and their salts with inorganic and/or organic bases, amino acids or amino acid amides are useful diagnostic and therapeutic agents.

Description

1 .. 94758

Macrocyclic compounds with 3,6,9,15-tetraazabicyclo [9.3. l] pentadecane structure, their use in compositions used for NMR, X-ray or radiodiagnostics and such compositions 5

The invention relates to 6-ring macrocyclic tetraacza complexing agents, to diagnostic compositions containing these compounds, to their use in diagnostic agents, and to methods for preparing these and diagnostic compositions.

Attention was paid to metal complexes as early as the early 1950s as radiological contrast agents. However, the compounds used in this case were so toxic that human use was out of the question. It was therefore surprising that certain complex salts have been shown to be so well tolerated that routine use in humans for diagnostic purposes could be considered. As a first representative of this class of compounds, the dimeglumine salt of Gd-DTPA [diethylenetriamine-20 pentaacetic acid gadolinium (III) complex] is described, which is described in EP Patent Application Publication No. 71,564 as a contrast agent in core spherical layer imaging. A weak point of this use is related to diseases of the central nervous system.

An essential rationale for the successful clinical use of Gd-DTPA is high efficiency in nuclear spindle layer imaging, especially in several brain tumors. Due to its good efficacy, Gd-DTPA at a dose of 0.1 mmol / kg body weight can be administered at significantly lower doses than, for example, X-ray contrast agents in many X-ray studies.

* As other representatives of the complex salts, the meglumine salt of Gd-DOTA [gadolinium (III) complex of 1,4,7,10-tetraazacyclododecanetraacetic acid] described in DE patent application 34 01 052 has proved its value for diagnostic purposes.

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However, the use of chelates at higher dosages would also be desirable. This applies in particular to the detection of certain diseases other than the central nervous system using nuclear-layer imaging (nmr diagnostics), and more particularly to the use of chelates as X-ray contrast agents.

In order to keep the volume load on the body as low as possible, it is necessary to use well-concentrated chelating solutions. The chelates known so far are very unsuitable for such use, above all because of their too high osmolality.

Therefore, there was a need to obtain chelates with lower osmolality than previously known chelates. At the same time, however, the conditions for the use of these compounds in humans should be maintained in relation to the difference (useful range), organ specificity, stability, contrast enhancing effect, tolerability and solubility between the effective dose of the complex compounds and the toxic dose in animal experiments.

The object of the invention was thus to provide these compounds and substances and to develop a method for their preparation which is as simple as possible. The present invention fulfills this function.

The complex compounds according to the invention and the solutions prepared therefrom surprisingly satisfy the said requirements. They have a lower osmolality and a useful range and / or stability of the chemical constituents of the solution as well as storage stability and / or organ specificity and / or a contrast enhancing effect (e.g.

30 relaxivity) and / or tolerability (e.g., less cardiovascular side effects or less hypersensitivity side effects) are more advantageous than the corresponding properties of previously available diagnostic agents.

Il 3. 94758

Likewise, without special measures, the pharmacokinetic properties of these compounds allow a better diagnosis of numerous diseases. The complexes remain and are excreted largely unchanged rapidly from the system, so that, especially with the use of relatively toxic metal ions, no harmful effects are observed despite the high dosage.

The use of these new complexes and complexing agents is in practice also facilitated by their advantageous coil stability.

A further advantage of the described complexes and complexing agents is that they are chemically very versatile. In addition to the choice of the central atom, their properties can be adjusted by selecting different subs-15 substituents on the macrocycle and / or different salt formers to meet the requirements for efficacy, pharmacokinetics, tolerability, solubility, workability, etc. Thus, for example, in diagnostics, the highly desirable specificity of the compounds for various structures of the body, specific biochemical substances, metabolic events, tissue or body fluid states can be achieved.

The macrocyclic compounds of the invention have the general formula (I) G

2b X

x ’X1

I I

ZOOC-CK-N N-CH-COOZ / T \ 30 / V. ' X c1 CH-X2. cooz where 35 ... denotes a single bond or a double bond, 4 94758 Q denotes a nitrogen atom or a group NH,

X1 and X2 independently of one another denote a hydrogen atom or a group - (CH2) n-OH or - (CH2) m- (CH) n -CH2OH, where n is one of the numbers 1 to 5 and OH

5 m is one of 0-2, A1, B1, C1 and D1 each independently represent a hydrogen atom or a group benzyloxymethyl or - (CH2) n-OH, where n is one of the numbers 1-5, G represents a hydrogen atom or another macrocycle which - - 10a is the general formula (II) Λ X1 X1 15 III (II), ZOOC-CH-M N-CH-COOZ ''

V

CH-X 2

20 COOZ

and Z represents a hydrogen atom and / or a meta-ionic equivalent of an element of the sequence numbers 21 to 29, 31, 32, 37 to 39, 42 to 44, 49 or 57 to 83, provided that X1 and X2 are only both hydrogen atoms when at least one of the four ring substituents A1, B1, C1 and D1 is other than a hydrogen atom.

Compounds of general formula (I) in which Z is a hydrogen atom are referred to as complexing agents, and are co-. Dysts in which at least two of the substituents Z are metal ion equivalents are called metal complexes.

The element according to the above-mentioned order, which forms the central ion of a physiologically acceptable complex salt, can, of course, also be radioactive for the desired use of the diagnostic substances according to the invention.

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If the compound of the invention is to be used in nmr diagnostics, the central ion of the complex salt must be paramagnetic. This is in particular the divalent and trivalent ions of the elements of the order numbers 21 to 29, 42, 44 and 58 to 70. Examples of suitable ions are chromium (III), manganese (II), iron (II), cobalt (II), nickel (II), copper (II), praseodymium (II), neodymium (II) , samarium (II) and ytterbium (III) ions. Due to their very strong magnetic moment, gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III) and iron (III) ions are particularly preferred.

If the compound of the invention is to be used in X-ray diagnostics, the central ion must be derived from an element of relatively high order in order to obtain adequate absorption of X-rays. Diagnostic agents containing a physiologically acceptable complex salt having a central ion of the elements of sequences 21 to 29, 42, 44, 57 to 83 were found to be suitable for this purpose; these include, for example, the lanthanum-20 (III) ion and the above-mentioned ions of the lanthanide series.

Preferred groups X1 are CH2OH, CH2CH2OH and CHOCH2OH and preferred groups X2 are CH2OH, CH2CH2OH, and CHOCH2OH.

The compounds of the invention have the desired properties described above. They contain the metal ions necessary for their use stably bound to the complex.

The osmolality level 30, which causes side effects such as pain, vascular damage and cardiovascular disorders, is significantly lower compared to Magnevist (Example Ib: 0.55 osmol / kg compared to 1.96 osmol / kg, 0.5 mol / liter 37 ° for Magnevist). C).

In MRI, the level of relativity as a measure of imaging is surprisingly high, so that the plasma sig-35 signal gain can be doubled compared to Magnevist, for example, when using the band of Example Ib 6-94758.

A further advantage of the present invention is that complexes containing hydrophilic or lipophilic substituents can now be prepared. This makes it possible to control the tolerability and pharmacokinetics of these complexes by chemical substitution.

The compounds of the invention can be coupled to appropriate bio- or macromolecules (see below for more details) to give complexes that show surprisingly high tissue and organ specificity.

The compounds of the invention are prepared from compounds of the general formula (I ') 15 Λ

,] f d'J

ZOOC-CH M N-CH-COOZ 'CH-X2 coor where l'. 2 '.1.2.

G ', X 3a X are G, X and X, respectively, in which the hydroxyl groups and functional groups contained therein are in protected form or as precursors, and Z' is a hydrogen atom or an acid protecting group, the protecting groups being cleaved off to give the general formula ( The complexing agents according to I) in which Z is hydrogen are reacted in a manner known per se with at least one metal oxide or metal salt formed from an element of the order numbers 21 to 29, 31, 32, 37 to 39, 42 to 44, 49 or 57 to 83, wherein Complexation can be performed before or after the hydroxyl groups.

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Suitable acid protecting groups Z 'are lower alkyl, aryl and aralkyl groups, for example methyl, ethyl, propyl, n-butyl, t-butyl, phenyl, benzyl, diphenylmethyl, triphenylmethyl, bis (p-5 nitrophenyl) methyl group, as well as trialkylsilyl groups.

Z 'may also denote an alkali metal.

The protecting groups are cleaved by methods known to those skilled in the art, for example by hydrolysis using hydrogenolysis, alkaline saponification of the ester with an alkaline substance in aqueous-alcoholic solution at 0-50 ° C, acidic saponification with inorganic acids, or in the case of tert-butyl esters with trifluoroacetic acid.

Suitable hydroxyl protecting groups are e.g.

15 benzyl, 4-methoxybenzyl, 4-nitrobenzyl, trityl, diphenylmethyl, trimethylsilyl, dimethyl-t-butylsilyl, diphenyl-t-butylsilyl group.

The hydroxyl groups can also be, for example, as THP-ether, α-alkoxyethyl ether, MEM-ether or ester formed with an aromatic or aliphatic carboxylic acid, such as, for example, acetic acid or benzoic acid. In the case of polyols, the hydroxyl groups can also be protected into ketals formed, for example, with acetone, acetaldehyde, cyclohexanone or benzaldehyde.

1 f. The hydroxyl groups present in 2 'X and X can also be protected by intramolecular esterification with the carboxyl groups in the α-position to the corresponding lactones.

Hydroxyl protecting groups can be removed by methods known to those skilled in the art, e.g., using hydrogenolysis, reductive cleavage with lithium / ammonia, treatment of ethers and ketals with acid or esters with an alkaline agent (see, e.g., 'Protective Groups in Organic Synthesis', TW Greene, 35 John Wiley & Sons, 1981).

3 94758

Dimeric compounds, i.e. compounds containing a second macrocycle of general formula (II), are synthesized by methods known from the literature, for example by an addition / removal reaction between an amine and a carbonyl compound (e.g. acid-5 chloride, mixed anhydride, activated ester, aldehyde).

Compounds of formula (I) in which G is a second macrocycle can also be synthesized by cyclizing tetra-halomethyl-4,4'-bis-pyridines (see below for more details).

Compounds of formula (I ') are synthesized by alkylation of compounds of general formula (III): a' 15 [i (III),

U -X N-U

</ \ 2 ° B C1

V

wherein U is hydrogen and V is an amino protecting group, or U is in each case an amino protecting group and V is hydrogen, wherein U and V may also be identical, for compounds of general formula (IV):

Nf-CH-COOZ '(IV) 11'

X

30 or with compounds of general formula (V):

Nf-CH-COOZ '(V) 1 2' ΧΔ wherein 35 Nf is a nucleofug such as Cl, Br, J, CH3-C6H4SO3, ch3so3, 4-NO2-C6H4SO3, cf3so3.

9. 94758 11 2 '.

The hydroxyl groups optionally included in the groups X and X may also, together with the OZ 'group, form a lactone.

Examples of alkylation reagents are: bromoacetic acid, chloroacetic acid, methyl bromoacetic acid, bromoacetic acid t-butyl ester, chloroacetic acid benzyl ester, 2-chloro-3-benzyloxypropanoic acid 2-bromo-2-bromo-2-bromobenzoic acid t-butyl ester (J. Gen. Chem. USSR 36 (1966) 52), 3,4-O-O-isopropylidene-2-p-tolylsulfonyl-3,4-dihydroxybutyric acid ethyl ester (Synth. Comm. 19 (1989) 3077 ), α-bromo-7-butyrolactone.

Examples of amino protecting groups U or V are: formyl, trifluoroacetate, benzoate, 4-nitrobenzoate, acetate, tosylate, mesylate, benzyl, 4-nitrobenzyl, 4-methoxybenzyl, trimethylsilyl, dimethyl-t-butylsilyl .

:. Alkylation of compounds of general formula (III) to compounds of general formula (I ') with compounds of general formula (IV) or (V) is carried out in polar, aprotic solvents such as, for example, dimethylformamide, acetonitrile, dimethylsulfoxide, aqueous tetrahydrofyde 25 or in hexamethylphosphoric acid triamide in the presence of an acid acceptor, which may be e.g. a tertiary amine [e.g. triethylamine, trimethylamine, N, N-dimethylaminopyridine, 1,5-diazabicyclo [4.3.0] -5-nonene (DBN), 1,5-diazabicyclo [5.4.0], 1,5-diazabicyclo [5.4.0 ] -5-un-decene (DBU)], alkali metal, alkaline earth metal carbonate or the like bicarbonate or hydroxide (e.g. sodium, lithium, magnesium, calcium, barium, potassium carbonate, hydroxide or bicarbonate), at a temperature of -10 - + 120 ° C, preferably 0-50 ° C, whereby catalytic amounts of an iodide or bromide may optionally be added.

_______ ___ I

10 94758

After cleavage of the remaining one or more amino protecting groups by methods known to those skilled in the art (e.g., acidic or basic hydrolysis, hydrogenolysis, reductive cleavage with alkali metals in liquid ammonia, reaction with tetrabutylammonium fluoride), the remaining one or more the amino function is reacted in a second alkylation reaction with a compound of formula (V) or (IV) to give compounds wherein X 1 is not the same as X 2.

The conversion of the desired 6-membered ring precursor included in the final product is performed by methods known to those skilled in the art. Examples are the hydrogenation of pyridine (Advan. Catal. 14 (1963) 203), the removal of oxygen from nitroxide rings (E. Klings-15 Berg, "The Chemistry of Heterocyclic Compounds", Volume 14, Part 2, Interscience Publishers, New York, 1961, p. 120).

The compounds of general formula (III) are synthesized by cyclization by methods known from the literature (e.g. Org. Synth. 58 (1978) 86); "Makrocyclic 20 Polyether Syntheses, Springer Verlag, Berlin, Heidelberg, New York, 1982; Coord. Chem. Rev. 3 (1968) 3; Ann. Chem. 1976 916; J. Org. Chem. 49 (1984) 110), wherein either of the two reactants used comprises two leaving groups at the end of the molecular chain and the other in turn two nitrogen atoms which nucleophilically displace these leaving groups.

An example is the reaction of A'-D'-substituted diethylenetriamines in which the nitrogen atoms at the chain end of these compounds displace nucleophilically leaving groups, e.g. 2,6-dihalomethylpyridines, 2,6-ditosylmethylpyridines or 2,6-dimers. -syylimetyylipyridiineistä. To synthesize dimeric compounds (i.e., G is a macrocycle), 2,2 ', 6,6'-tetrachloromethyl-4,4'-bis-pyridine 35 is used in the cyclization reaction (see, e.g., Synthesis 1989 552).

11 94758

The nitrogen atoms are optionally protected (e.g. to tosylates or trifluoroacetates) and are liberated * prior to the subsequent alkylation reaction by methods known in the art (tosylates e.g.

5 using inorganic acids, alkali metals in liquid ammonia, hydrobromic acid and phenol product,

RedAl, lithium aluminum hydride, sodium amalgam, cf. e.g., Liebigs Ann. Chem. 1977 1344; Tetrahedron Letters 1976 3477; trifluoroacetates, e.g. using inorganic acids or ammonia in methanol, cf. e.g., Tetrahedron Letters 1967 289).

To prepare macrocycles which are variously substituted on nitrogen atoms, these atoms in the compounds of formula (I) may be provided with various protecting groups, e.g. tosylate and benzyl groups. The latter are then also removed by methods known from the literature (preferably by hydrogenation, e.g. EP patent application 232,751).

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

Correspondingly substituted bis-aldehydes or bis-ketones can also be cyclized, e.g.

2,6-bis-acetylpyridine, in each case with the desired end positions with the bis-amines, after which the Schiff bases thus obtained are reduced by methods known from the literature, e.g. by catalytic hydrogenation (Helv. Chim. Acta 61 (1978) 1376).

The amines required as starting materials for the cyclization are prepared analogously to methods known from the literature (e.g. EP 299 795).

Using an N-protected amino acid as a starting material, the reaction with a partially protected diamine 35 (e.g. by the carbodiimide method) gives the triamine after cleavage of the protecting groups and reduction with diborane.

94758

The hydroxyl and carboxyl groups of the compounds of formula (I) may be converted into a suitable functional group for attachment to a macro- or biomolecule by methods known to those skilled in the art (Chem. Pharm. Bull. 31 (1985) 674; "Compendium of

Organic Synthesis ", parts 1-5, Wiley & Sons, Inc.; Houben-Weyl," Methoden der Organischen Chemie ", part VIII, Georg Thieme Verlag, Stuttgart, J. Biochem. 92 (1982) 1413) as desired substituents (e.g. using amino, hydrazino, hydrazinocarbonyl, epoxide, anhydride, methacryloylhydrazinocarbonyl, maleimidamidocarbonyl, halogen, halocarbonyl, mercapto, isothiocyanate functional groups).

Examples of the conversion of bonded hydroxyl groups are their reactions in suitable solvents such as tetrahydrofuran, dimethoxyethane or dimethyl sulfoxide, 2-phase aqueous systems such as water / dichloromethane, an acid acceptor such as sodium hydroxide, sodium hydride or alkali metal alkali metal or alkali metal alkali metal or alkali metal poly (4-vinylpyridine) -Reillex, rium, magnesium, potassium, calcium carbonate or damping, in the presence of a temperature from 0 ° C to the boiling point of the respective solvent, but preferably at a temperature of 20 to 60 ° C, with the substrate, having the general formula (VII)

Nf-L-Fu (VII) wherein L is an aliphatic, aromatic, aryl aliphatic, branched, straight-chain or cyclic hydrocarbon residue containing up to 20 carbon atoms, and Fu is a desired end-functional group optionally protected (DE Application Publication 34 17 413).

„94758

Examples of compounds of general formula (VII) are Br (CH2) 2NH2, Br (CH2) 3OH, BrCH2COOCH3,

Br CH2CO2-t-Bu, C1CH2CONHNH2, Br (CH2) 4CO2C2H5, BrCH2COBr, 0/5 BrCH2CONH2, C1CH2C00C2H5, BrCH2CONHNH2, BrCH2-CH-CH2, CF3SO3 (CH2) 3Br, BrCH2C = CH, BrCHCH

Conversions of carboxyl groups can be performed, for example, by the carbodiimide method (Fieser, "Reagents for Organic Syntheses, Vol. 10, p. 142) using 10 mixed anhydrides (Org. Prep. Proc. Int. 1_ (1975) 215) or an activated ester ( Adv. Org. Chem., Part B, p. 472).

The complexing ligands (as well as complexes) thus obtained can also be coupled to bio- or macromolecules which are known to accumulate in particular in the organ or part of the organ under study. Such molecules include, for example, enzymes, hormones, polysaccharides such as dextrans or starches, porphyrins, bleomycin, insulin, prostaglandins, steroid hormones, amino sugars, amino acids, peptides such as polylysine, antibodies to proteins such as substances, lectins), lipids (including as liposomes) and nucleotides of the DNA or RNA type. Particular mention may be made of conjugates formed with albumins, such as human serum albumin, with antibodies, such as monoclonal antibodies, with antibodies specific for tumor-associated antigens or with antimyosin. Instead of biological macromolecules, suitable synthetic polymers such as polyethyleneimines, poly-amides, polyureas, polyethers such as polyethylene glycols and polythiureas can also be coupled. The pharmaceutical agents thus formed are suitable for use in, for example, tumors! infarktidiagnostiikassa. Monoclonal antibodies (see, for example, Nature 256 (1975) 495) have the advantage over polyclonal antibodies that monoclonal antibodies are specific for an antigenic determinant, have a certain binding affinity, they are homogeneous (thus substantially simplifying obtaining them pure) and can be prepared in large quantities in 5 cell cultures. Suitable for tumor screening, for example, are monoclonal antibodies or fragments Fab and F (ab ') 2 / which are specific for, for example, tumors of the human stomach and small intestine, breast, liver, bladder, gonads and melanoma 10 (Cancer Treatment Repts. 68 (1984)). 317; Bio Sci 34 (1984) 150) or target carcinomybronial antigen (CEA), human choriogonadotropin (B-HCG) or other tumor-associated antigens such as glycoproteins (New Engl. J. Med. 298 (1973) 1384; U.S. Patent 4,331,647) to 15. Suitable are e.g. also antimyosin, anti-insulin, and antifibrin antibodies (U.S. Patent 4,036,945).

Colon cancers can be detected nmr diagnostically using gadolinium (III) ion complexed: conjugates with antibody 17-1A (Centocor, USA).

Conjugates or conjugates formed with liposomes using, for example, unilamellar or multilamellar phosphatidylcholine cholesterol vesicles are suitable for liver research or for tumor diagnosis.

In the case of antibody conjugates, binding of the antibody to the complex or ligands must not result in loss or reduction of the binding affinity and binding specificity of the antibody for the antigen. This can be accomplished by performing binding to either the carbohydrate moiety in the Fc portion of the glycoprotein 30 or to Fab or F (ab ') 2 fragments or to sulfur atoms of the antibody or antibody fragments.

In the first case, oxidative cleavage of the sugar units must first be performed to form the coupling formyl groups. This oxidation can be carried out chemically using oxidizing agents such as, for example, periodic acid, sodium metaperiodate or potassium metaperiodate according to methods known from the literature (see, for example, J. Histochem. And Cytochem. 22. (1974) 5 1084) in aqueous solution and concentrations of 1 to 100, preferably 1-20 mg / ml, as well as an oxidant concentration of 0.001 to 10 mM, preferably 1 to 10 mM, at a pH of about 4 to 8 and a temperature of 0 to 37 ° C and a reaction time of 15 minutes to 24 hours. The oxidation can also be carried out enzymatically, for example using galactose oxidase, an enzyme concentration of 10 to 100 units / ml, a substrate concentration of 1 to 20 mg / ml, a pH of 5 to 8 and a temperature of 20 to 40 ° C, and a reaction time of 1 to 8 hours (see for example, J. Biol. Chem. 234 (1959) 445).

Complexes or ligands with suitable functional groups, such as hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide, are bound to the aldehydes formed by oxidation, the reaction being carried out at 0 to 37 ° C for 1 hour, the reaction is allowed to proceed for 1 hour. over time, the pH is about 5.5 to 8, the antibody concentration is 0.5 to 20 mg / ml, and the molar ratio of complexing agent to antibody aldehydes is 1: 1 to 1,000: 1. Stabilization of the conjugate is then performed by reduction of the double bond, e.g.

Sodium borohydride or sodium cyanoborohydride, in which case a 10 to 100-fold excess of reducing agent is used (see, for example, J. Biol. Chem. 254 (1979) 4359).

Another possibility for forming antibody conjugates is based on the protective reduction of disulfide bridges of the iramunoglobulin molecule 30, in which the more sensitive disulfide bridges between the H chains of the antibody molecule are cleaved, while the SS bonds in the antigen-binding region remain unaffected, occur virtually not at all (Biochem. 18 16 94758 (1979) 2226; Handbook of Experimental Immunology, Part 1, 2nd Edition, Blackwell Scientific Publications, London, 1973, Chapter 10). These free sulfhydryl groups in the H-chain end regions are then reacted with the appropriate functional groups in complexing agents or metal complexes at 0-37 ° C at a pH of about 4-7 to give the reaction in 3-72 hours and a covalent bond is formed. which does not affect the antigen binding region of the antibody. Suitable reactive groups include, for example: haloalkyl, haloacetyl, p-mercurobenzoate, isothiocyanate, thiol, epoxide groups and groups which react in the Michael addition reaction, such as, for example, maleimide, methacrylic groups. (e.g. J. Amer. Chem. Soc. 101 (1979) 15 3097).

In addition, a number of suitable, often commercially available, bifunctional "linkers" (see, for example, Pierce, Handbook and General 20 Catalog, 1986) that react with both SH groups and complexes of fragments to find antibody fragments for attachment to complexes or ligands are found. with amino or hydrazino groups.

Examples are: 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-pyridyldi-30-thio) propionic acid ester (SDPD); 4- [3- (2,5-dioxo-3-pyrrolinyl) propionyloxy] -3-oxo-2,5-diphenyl-2,3-dihydrothiophene-1,1-dioxide; asetyylialanyylileusyylialanyyli-aminobenzyl; acetamido-p-tioureidobentsyyli.

Non-covalent bonds of the 35 type can also be used in the coupling, in which case both ionic and van 17 94758 der Waals bonds and hydrogen bridge bonds can contribute to the bond in varying proportions and strengths (key-lock principle) (e.g. avidin-biotin, antibody-antigen ). Fusion compounds of smaller complexes ("host-foreign") in larger cavities of the macromolecule are also possible.

The coupling principle is to first prepare a bi-functional macromolecule by either fusing an anti-tumor antigen antibody hybridoma to another antibody hybridoma against the complex of the invention or chemically linking the two antibodies via a linker (e.g., as shown in in J. Amer. Chem. Soc. 101 (1979) 3097) or an antibody against a tumor antigen is optionally linked via a linker to avidin (e.g. biotin) (DJ Hnatowich et al., J. Nucl. Med. 28 (1987)). ) 1294). Instead of an antibody, its corresponding Fab or F (ab ') 2 fragments can also be used. In pharmaceutical use, a bifunctional macromolecule that is enriched in the target region is injected first, and then after a period of time a complex compound of the invention [optionally bound to biotin (or avidin)] that binds to the target region in vivo and has a diagnostic effect here.

25 In addition, the use of other coupling methods may be considered, such as in Protein Tailoring Food Med. Uses, Am. Chem. Soc. Symp. 1985 349, use of the "reverse radiolabel" described.

With so-called solid phase coupling, a particularly simple method is provided for the preparation of antibody conjugates or antibody fragment conjugates: the antibody is coupled to a stationary phase (e.g. an ion exchanger) in a glass column, for example. Rinsing the column several times with a solution suitable for aldehyde group formation, washing, rinsing with a solution containing the functionalized complex, and finally eluting the conjugate gives very high conjugate yields.

With this method, it is possible to automatically and continuously produce the desired amounts of conjugate.

Other coupling steps can also be implemented in this way.

Thus, for example, fragment conjugates can be prepared with the sequence papain-pel-reduction / bifunctional linker / functionalized complex or ligand.

The compounds thus formed are then preferably purified by ion exchange chromatography in a flash protein-liquid chromatography apparatus.

The metal complexes according to the invention are prepared in the manner disclosed in DE-A-34 01 052, in which a metal oxide or a metal salt (for example nitrate, acetate, carbonate, chloride or sulphate) of the elements 21 to 29, 42, 44, 57 to 83 is dissolved or slurrying in water and / or a lower alcohol (such as methanol, ethanol or isopropanol), and reacting the solution or slurry with an equivalent amount of the complexing ligand solution or slurry, followed by replacing the acidic hydrogen atoms present with inorganic and / or organic bases or amino acids, if desired. cations.

The desired metal ions can then be incorporated just as well before or after cleavage of the protecting groups of the hydroxyl groups and functional groups as before or before or after the formation and binding of the functional groups to the macro- or biomolecule.

The diagnostic compositions according to the invention are likewise prepared in a manner known per se, in which case the complex compounds according to the invention - to which may be added in pharmaceutical preparations conventionally

II

94758 19 used additives - slurry or dissolve in an aqueous medium, after which the slurry or solution may be sterilized. Suitable additives are, for example, physiologically innocuous buffers (such as tromethamine, for example), complexing additives (such as diethylenetriaminepentaacetic acid) or, if appropriate, electrolytes, such as sodium chloride, or, if necessary, antioxidants, such as ascorbic acid.

If it is desired to prepare slurries or solutions of the substances of the invention in water or physiological saline to obtain an enteric diagnostic composition or for other reasons, they are mixed with one or more excipients customary in pharmaceutical preparations (for example methylcellulose, lactose, mannitol) and / or surfactant. , Tween®, Myrj®) and / or a flavoring agent (e.g. essential oils).

: In principle, it is also possible to prepare the diagnostic compositions according to the invention without isolating the complex salts. In any case, special attention should be paid to the fact that the chelation is carried out in such a way that the salts and saline solutions according to the invention are practically free of non-complexed, toxic metal ions.

This can be confirmed, for example, by using color indicators such as xylenol orange in control titrations during the manufacturing process. The final confirmation is the purification of the isolated complex salt.

The cowardly diagnostic compositions of the invention preferably contain 0.1 μmol to 1 mol / liter of complex salt and are generally administered in amounts of 0.1 μmol / 5 mmol / kg. They are suitable for internal and external use in the digestive tract. The complex compounds 35 of the invention may be used: 1. 94758 in a nmr and X-ray diagnostic droplet complexed with ions of the elements of order numbers 21-29, 42, 44 and 57-83; 2. in radiodiagnostics in complex with radioisotopes which are isotopes of the elements of the order numbers 27, 29, 31, 32, 37-39, 43, 49, 62, 64, 70, 75 and 77.

The diagnostic agents of the invention meet the various conditions set for suitability as a contrast agent for core-sphere deposition. Thus, when administered orally or parenterally, they are very well suited for increasing the signal intensity to improve the authenticity of the image obtained in nuclear spin layer imaging. Furthermore, they have the high efficiency necessary to load the body with the lowest possible amounts of foreign substances and the good tolerability necessary to maintain the non-invasive nature of the studies.

Thanks to the good water solubility and low osmolality of the diagnostic substances according to the invention, it is possible to prepare well-concentrated solutions, whereby the circulating volume load can be kept within acceptable limits and the dilution caused by body fluid must be equalized, i.e. 25 nmr diagnostic substances must be 100-1000 times more water-soluble than diagnostic agents for nmr spectroscopy. Furthermore, the drugs according to the invention not only show good stability in vitro, but also show a surprisingly high stability in vivo, whereby non-covalently bound - inherently toxic - ions of the complex are released or exchanged only extremely slowly during the time taken until these again, the new contrast agents have been completely eliminated through secretion.

94758 21

In general, the substances according to the invention are administered for use in nmr diagnostics in amounts of 0.0001 to 5 mmol / kg, preferably 0.005 to 0.5 mmol / kg. Details of use are discussed, for example, in H.J. Weinmann 5 et al., Am. J. of Roentgenology 142 (1984) 619.

Particularly low doses (less than 1 mg / kg body weight) of organ-specific nmr diagnostic agents can be used, for example, to detect tumors and myocardial infarction. Furthermore, the complex compounds of the invention can be advantageously used as sensitivity reagents and transfer reagents in nmr spectroscopy in vivo.

Due to their advantageous radioactive properties and the good stability of the complex compounds contained therein, the substances according to the invention are also suitable as radiodiagnostic substances. Details of their use and administration are described, e.g., in "Radiotracers for Medical Applications", CRC-Press, Boca Raton, Florida.

Another imaging method using radioisotope-20s is positron emission layer imaging using positron emitting isotopes such as e.g.

43Sc, "Se, 52Fe, 55Co and 68Ga (Heiss, W.D., Phelps, M.E.," Positron Emission Tomography of the Brain ", Springer Verlag,

Berlin, Heidelberg, New York, 1983).

The diagnostic agents according to the invention are very well suited as X-ray contrast agents, in which case it should be emphasized in particular that no signs of the reactions of the nature of severe hypersensitivity reactions known in connection with iodinated contrast agents can be observed in biochemical pharmacological studies.

These substances are particularly valuable due to their advantageous absorption properties in the range of higher tube voltages in digital substration techniques.

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

Details of the use of X-ray contrast agents are discussed, for example, in Barke, "X-Ray Contrast-5 Mittel", G. Thieme, Leipzig, 1970; and P. Thurn, E. Buech-eler, "Einfuehrung in die X-ray Diagnostics", G. Thieme, Stuttgart, New York, 1977.

All in all, new complexing agents, metal complexes and metal complex salts have been synthesized, which offer new possibilities in diagnostics. Above all, the development of new types of imaging methods for medical diagnostics makes this development seem desirable.

The following examples are intended to illustrate the subject matter of the invention in more detail.

Example 1 a) 3 / 6,9-Tris [dihydro-2 (3H) -furanon-3-yl] -3,6,9 / 15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11 , 13-triene 20 To a mixture of 10 g (48.48 mmol) of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene, 33.5 g (242.38 mmol) of potassium carbonate and 805 mg (4.85 mmol) of potassium iodide in 200 ml of acetonitrile, 40 g (242.38 mmol) of α-bromo-7-butyrolactone are added. This mixture is refluxed for 48 hours, then evaporated to dryness in vacuo. The residue is slurried in 500 ml of methylene chloride and the slurry is extracted 3 times with 150 ml of water. The organic phase is dried over magnesium sulphate and evaporated to dryness in vacuo. The residue is purified by chromatography on silica gel (eluent: methylene chloride / methanol, 15: 1) to give a yield of 7.11 g (32% of theory) of a pale yellow oil which solidifies on standing.

Analysis: 35 C 60.25; H 6.59; N 12.22 (calc.) C 60.18; H 6.64; N 12.17 (tod.).

Il 94758 23

L

b) 3,6 / 9/15-tetraazabicyclo [9.3.1] pentadeca-1 (15) / 11/13-triene-3/6/9-tris [α- (2-hydroxyethyl) acetic acid] gadolinium complex 6.7 g (14.61 mmol) of the title compound 5 of Example 1a are dissolved in 50 ml of deionized water and the pH of the solution is adjusted to 5.5 by adding 1N hydrochloric acid. 2.65 g (7.3 mmol) of gadolinium oxide are added to the solution, after which the mixture is refluxed for 3 hours. The cooled solution is stirred for 1 hour in a flow 10 roll with 10 ral of acidic ion exchanger (IR 120) and 10 ml of basic ion exchanger (IRA 410). The ion exchanger is filtered off the solution and the filtrate is boiled for 1 hour with activated carbon. Filtration and freeze-drying give 9.25 g (95% of theory) of an amorphous, colorless powder (containing 8.3% of water by analysis).

Analysis (corrected for water present): C, 41.43; H 4.99; N 8.40; Gd 23.58 (calc.) C 41.35; H 5.09; N 8.34; Gd 23.50 (tod.).

c) 3 / 6,9,15-Tetraazabicyclo [9.3.1] pentadeca-20 1 (15), 11,13-triene-3,6,9-tris [α- (2-hydroxyethyl) acetic acid ] europium complex

In analogy to the above, the use of the compound Is1E2O3 as a starting material gives the corresponding europium complex.

Analysis (corrected for water present): 25 C 41.76; H 5.03; N 8.47; EU 22.97 (calc.) C 41.68; H 5.12; N 8.39; Eu 22.88 (tod.).

Example 2 a) 3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris [α- (benzyloxymethyl) -30 acetic acid]

A solution of 10 g (48.48 mmol) of 3,6,9,15-tetraazabicyclo [9. 3.1] pentadeca-1 (15), 11,13-triene and 114.71 g (484.8 mmol) of the sodium salt of 2-chloro-3-benzyloxypropionic acid in 200 rol of water are heated at 70 ° for 48 hours 35 hours. C. The solution is diluted with 400 ml of hydrogen and then 300 ml of 2N hydrochloric acid are added. The mixture is extracted 5 times with 200 ml of methylene chloride each time. The aqueous phase is separated and evaporated to dryness in vacuo. The residue is dissolved in 300 ml of ethanol 5 and filtered off from sodium chloride. The solution is evaporated to dryness in vacuo and the residual oil is chromatographed on silica gel (eluent: ethanol / water, 20: 1). The main fractions are combined, evaporated to dryness in vacuo and the residue is dissolved in 50 ml of 5% hydrochloric acid solution. The solution is loaded onto a column which is. ® packed with Reillex (= poly-4-vinylpyridine) and the product is eluted from the column with a 3: 1 mixture of water and methanol. The main fractions are combined and evaporated to dryness to give 12.93 g (36% of theory) of a strongly hygroscopic solid (containing 9.1% of water by analysis).

Analysis (corrected for water present): C, 66.47; H 6.53; N 7.56 (calc.) C 66.38; H 6.60; N 7.48 (tod.).

20 b) 3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-1 (15), 1,3-triene-3,6,9-tris [α- (hydroxymethyl) acetic acid] 12 .6 g (17.01 mmol) of the title compound of Example 2a are dissolved in a mixture of 200 ml of methanol and 100 ml of water and 4 g of palladium catalyst (10% palladium (Pd) on activated carbon) are added to the solution. The mixture is hydrogenated for 5 hours at 50 [deg.] C., after which the catalyst is filtered off and the solution is evaporated to dryness in vacuo.

Yield: 7.84 g (98% of theory) of glassy solid (containing 6.9% of water by analysis).

Analysis (corrected for water present): C, 51.06; H 6.43; N 11.91 (calc.) C 50.97; H 6.51; N 11.81 (tod.).

C) 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris [α- (hydroxymethyl) acetic acid] gadolinium complex 7.5 g (15.94 mmol) of the title compound 5 of Example 2b are dissolved in 50 ml of deionized water and 2.89 g (7.97 mol) of gadolinium oxide are added to the solution. The solution is heated at 90 ° C for 3 hours. The cooled solution is stirred at room temperature for 1 hour alternately with 2 ml of acidic ion exchanger (IR 120) and 2 ml of basic ion exchanger (IRA 410). The ion exchanger is filtered off the solution and the filtrate is boiled briefly with activated carbon. Filtration and freeze-drying give 9.56 g (96% of theory) of an amorphous, colorless powder (containing 8.1% of water by analysis).

Analysis (corrected for water present): C, 38.45; H 4.36; N 8.97; Gd 25.17 (calc.) C 38.37; H 4.43; N 8.89; Gd 25.06 (tod.).

Example 3 a) 3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-20 1 (15), 11,13-triene-3,6,9-tris [α- (1,2-O -isopropylidene-1,2-dihydroxyethyl) acetic acid ethyl ester]

A mixture of 15 g (72.71 mmol) of 3,6,9,15-tetraazabicyclo (9.3.1] pentadeca-1 (15), 11,13-triene, 156.38 g (436.28 mmol) ) 3,4-O-Isopropylidene-2- (p-tolylsulfonyl-2H) -3,4-dihydroxybutyric acid ethyl ester, 60.3 g (436.28 mmol) of potassium carbonate and 2.41 g (14.54 mmol) of potassium ion. -dide in 400 ml of acetonitrile, refluxed for 48 hours, evaporated to dryness in vacuo and the residue slurried in 500 ml of methylene chloride, the slurry is extracted 3 times with 200 ml of water and the organic phase is dried over magnesium sulphate and evaporated. dryness.

The residual oil is chromatographed on silica gel (eluent: methylene chloride / hexane / methanol, 20: 4: 1).

Yield: 17.24 g (31% of theory) of a yellow, 35 viscous oil.

26 94758

Analysis: C, 59.67; H 7.91; N 7.32 (calc.) C 59.59; H 7.98; N 7.27 (tod.).

b) 3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-5 1 (15), 11,13-triene-3,6,9-tris [α- (1,2-dihydroxyethyl) - acetic acid] 16.5 g (21.57 mmol) of the title compound of Example 3a are dissolved in 100 ml of ethanol and 50 ml of 5N sodium hydroxide solution are added to the solution. The mixture is refluxed for 10 hours, then evaporated to dryness in vacuo. The residue is dissolved in 250 ml of methanol and filtered off from sodium chloride. The filtrate is evaporated to dryness in vacuo and the residue is purified by ion exchange as follows: it is dissolved in 50 ml of water and the solution is loaded onto a cation exchange column (IR 120). The column is rinsed with water, after which the ligand is eluted with 0.5 N aqueous ammonia. The main fractions are combined, evaporated to dryness, the residue is slurried in a small amount of water and the slurry is loaded onto an ion exchange column 20 (IRA 67). The column is washed first with water and then eluted with 0.5 N formic acid solution. The desired eluate is evaporated to dryness in vacuo and the residue is dissolved in a small amount of hot methanol. Carefully add acetone and cool in an ice bath to crystallize the ot-25 silica compound.

Yield 8.22 g (68% of theory) of a glassy solid (containing 9.2% of water by analysis).

Analysis (corrected for water present): C, 49.28; H 6.47; N 9.99 (calc.) 30 C 49.17; H 6.56; N 9.88 (tod.).

* c) 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris [e- (1,2-dihydroxyethyl) - acetic acid] gadolinium complex 8 g (14.27 mmol) of the title compound 35 of Example 3b are dissolved in 60 ml of deionized water and 2.58 g (7.135 mmol) of gadolinium oxide are added to a solution of 2V 94758. The mixture is heated for 3 hours at 90 ° C, after which the cooled solution is stirred at room temperature for 1 hour alternately with 2 ml of acidic ion exchanger (IR 120) and 5 ml of basic ion exchanger (IRA 410). The ion exchanger is filtered off the solution and the filtrate is boiled with activated carbon. Filtration and freeze-drying give 9.89 g (97% of theory) of an amorphous, colorless powder (containing 7.3% of water by analysis).

Analysis (corrected for water present): C 38.65; H 4.65; N 7.84; Gd 22.00 (calc.) C 38.54; H 4.74; N 7.78; Gd 21.92 (tod.).

Example 4 a) 2,2 ', 6 / 6'-Tetra (hydroxymethyl) -4,4'-bipyridine 50 g (128.77 mmol) of 2,2', 6,6'-tetra (methoxycarboxylate) nyl) -4,4'-bipyridine is dissolved in a mixture of 400 ml of dioxane and 400 ml of water and 48.71 g (1.28 mol) of sodium borohydride are added in small portions. The mixture is stirred overnight at room temperature. The solution is acidified by adding 5 N hydrochloric acid, after which it is evaporated to dryness. The residue is slurried in 1 liter of 1 N sodium hydroxide solution and the slurry is extracted 3 times with 250 ml of chloroform. The organic phase is dried over magnesium sulphate and evaporated to dryness in vacuo.

The residue is recrystallized from ethanol / ether.

Yield: 29.53 g (83% of theory) of colorless crystals.

Analysis: 30 C 60.86; H 5.84; N 10.14 (calc.): C 60.77; H 5.93; N 10.06 (tod.).

b) 2,2 ', 6,6'-Tetra (chloromethyl) -4,4'-bipyridine 29 g (104.96 mmol) of the title compound of Example 4a are refluxed in 250 g (2.1 mol) of thionyl chloride. For 5 hours. The mixture is evaporated to dryness and 28. 9 4 7 5 8 The residue is slurried in 200 ml of concentrated soda solution. The mixture is extracted twice with 150 ml of methylene chloride. The organic phase is dried over magnesium sulphate and evaporated to dryness in vacuo. The residue is crystallized from ether / hex-5.

Yield: 35.54 g (94% of theory) of colorless crystals.

Analysis: C 48.03; H 3.45; N 8.08; Gd 40.51 (calc.) 10 C 48.10; H 3.40; N 7.96; Gd 40.59 (tod.).

c) 13,13'-bis- [3,6,9-tris (p-tolylsulfonyl) -3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13- triene] to 118.43 g (194.25 mmol) of N, N ', N "-tris (p-tolyl-sulfonyl) diethylenetriamine N, N" disodium salt in 1600 ml of dimethylformamide are added dropwise over 4 hours At 100 ° C, a solution of 34 g (97.12 mmol) of the title compound of Example 4b (dissolved in 700 ml of dimethylformamide). The mixture is stirred overnight at 100 ° C. To the Moon-20 earth solution is added dropwise 2 liters of water and the mixture is cooled to 0 ° C. The precipitate is filtered off with suction and washed with water. After drying in vacuo (60 ° C), the precipitate is recrystallized from acetonitrile to give 79.13 g (61% of theory) of a cream powder.

Analysis: C 57.55; H 5.28; N 8.39; S 14.40 (calc.) C 57.47; H 5.35; N 8.31; S 14.32 (tod.).

d) 13,13'-Bis- [3,6,9,15-tetraazabicyclo [9.3.1] -pentadeca-1 (15), 11,13-triene] octahydrosulphate 79 g (59.15 mmol) ) of the title compound * of Example 4c is dissolved in 270 ml of concentrated sulfuric acid and the solution is stirred for 48 hours at 100 ° C. The solution is cooled to 0 ° C and 1.35 liters of absolute ether are added dropwise. The precipitate is filtered off with suction and mixed with 500 ml of methanol. Filtration and vacuum drying on a 29 94758 dowel gives 65.74 g (93% of theory) of an air-meltable solid.

Analysis: C 22.11; H 4.22; N 9.38; S 21.46 (calc.) Δ C 22.04; H 4.33; N 9.29; S 21.38 (tod.).

e) 13,13'-bis- [3,6,9,15-tetraazabicyclo [9.3.1] -pentadeca-1 (15), 11,13-triene] 65.5 g (54.80 mmol) the title compound of Example 4d is dissolved in 100 ml of water and the pH of the solution is adjusted to 13-10 by adding 32% sodium hydroxide solution. The mixture is extracted 3 times with 250 ml of hot toluene. The toluene phases are separated and combined, then they are refluxed for 1 hour with 20 g of finely ground sodium hydroxide. The mixture is filtered and the filtrate is evaporated to dryness.

Yield: 21.6 g (96% of theory) of a pale yellow solid.

Analysis: C 64.36; H 8.35; N 27.29 (calc.) 20 C 64.27; H 8.44; N 27.22 (tod.).

f) 13,13'-bis- [3,6,9,15-tetraazabicyclo [9.3.1] -pentadeca-1 (15), 11,13-triene-3,6,9-tris- [a - (benzyloxy-carboxymethyl) acetic acid]

A mixture of 21.5 g (52.37 mmol) of the title compound of Example 4e: 25 and 247.8 g (1.05 mol) of the sodium salt of 2-chloro-3-benzyloxypropionic acid in 400 ml of water is heated for 48 hours. At 70 ° C. The solution is diluted with 800 ml of water and then 600 ml of 2N hydrochloric acid are added. The mixture is extracted 5 times with 300 ml of methylene chloride each time. The aqueous phase is evaporated to dryness in vacuo. The residue is dissolved in 500 ml of ethanol and filtered off from sodium chloride. The solution is evaporated to dryness in vacuo and the residue is chromatographed on silica gel (eluent: ethanol / water, 20: 1). The main fractions 35 are combined, evaporated to dryness in vacuo and the residue 30. 9 4 7 5 8 is dissolved in 100 ml of 5% hydrochloric acid solution.

. ... ®

The solution is loaded onto a column packed with Reillex (= poly-4-vinylpyridine) and the product is eluted from the column with a 2: 1 mixture of water and methanol. The main fractions were combined and evaporated to dryness to give 20.92 g (27% of theory) of a highly hygroscopic solid (8.1% water by analysis).

Analysis (corrected for water present): C, 66.56; H 6.40; N 7.57 (calc.) 10 C 66.47; H 6.51; N 7.48 (tod.).

g) 13,13'-bis- [3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris- [a - (hydroxymethyl) acetic acid] 20.5 g (13.85 mmol) of the title compound 15 of Example 4f are dissolved in a mixture of 300 ml of methanol and 150 ml of water, and 7 g of palladium catalyst (10% palladium on activated carbon) are added to the solution. The mixture is hydrogenated for 5 hours at 50 ° C. The catalyst is filtered off and the solution is evaporated to dryness in vacuo.

Yield: 12.62 g (97% of theory) of a glassy solid (8.5% of water by analysis).

Analysis (corrected for water present): C 51.17; H 6.23; N 11.93 (calc.) C 51.07; H 6.31; N 11.87 (tod.).

H) 13,13'-bis- [3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris- [ α- (hydroxymethyl) acetic acid] gadolinium complex 12 g (12.78 mmol) of the title compound of Example 4g are dissolved in 80 ml of deionized water and 4.63 g (12.78 mmol) of gadolinium oxide are added to the solution. The mixture is heated at 90 ° C for 3 hours. The saturated solution is stirred for 1 hour at room temperature alternately with 5 ml of acidic ion exchanger (IR 120) and 5 ml of basic ion exchanger (IRA 410). The ion exchanger is filtered off and the filtrate is boiled briefly with activated carbon 3i 94758. Filtration and freeze-drying give 15.3 g (96% of theory) of a colorless, amorphous powder (containing 9.3% of water by analysis).

Analysis (corrected for water present): δ C 38.52; H 4.20; N 8.98; Gd 25.21 (calc.) C 38.46; H 4.28; N 8.91; Gd 25.14 (tod.).

Example 5 a) Trans-5- (p-tolylsulfonyl) amino-6- (p-tolylsulfonyloxy) -2,2-dimethyl-1,3-dioxepane In a solution of 100 g of trans-6-amino 2,3-Dimethyl-1,3-dioxepan-5-ol in 903 ml of pyridine, 295.67 g of p-toluenesulphochloride are added in small portions while stirring at -5 to 0 ° C. The mixture is left for 72 hours at + 4 ° C, after which it is stirred in 10 liters of 15 ml of ice water. The mixture is filtered off with suction and the precipitate obtained is washed with water, after which the residue is dried in an oven at 50 ° C under a pressure of 200 torr for 48 hours. To purify the obtained crude product, it is recrystallized from 5 liters of dioxane to give 196 g of the title compound-20 as a white powder.

Sp. 200-202 ° C.

b) K- [2- (N-tolylsulfonylamino) ethyl] -p-tolylsulfonamide monosodium salt 150 g of N- [2- (N-tolylsulfonylamino) ethyl] -p-to-25-sulfonylamide are slurried in 1.25 liters of ethanol. is refluxed and a solution of 10.3 g of sodium in 300 ml of ethanol is added dropwise to give a solution. As the solution cools, the title compound precipitates. It is filtered off with suction, washed with ethanol and dried at 50 [deg.] C. and 200 torr to give 119 g of the title compound as a white powder.

32 94758 c) Cis-2,2-Dimethyl-5- [N- (p-tolylsulfonyl) amino] -6- [N- (p-tolylsulfonyl) -N- (Ν'-2-p-tolylsulfonylaminomethyl) )] - 1,3-dioxepane 116 g of the monosodium salt of Example 5b are poured into 2.66 liters of dimethylformamide. A solution of 141 g of trans-5- (p-tolylsulfonyl) amino-6- (p-tolylsulfonyloxy) -2,2-dimethyl-1,3-dioxepane in 1.5 liters is added dropwise to the slurry at 100 ° C. dimethylformamide, and the mixture is stirred for 5 hours at a bath temperature of 120 ° C. The reaction solution is then evaporated in vacuo to 1 liter and diluted by adding 10 liters of ice water. The precipitate formed is filtered off with suction, washed with water and dried at 50 ° C and 200 torr to give 182 g of the crude title compound, which is boiled in 1.85 liters of ethanol to purify it. The precipitate is filtered off with suction and dried to give 125 g of the title compound as a white powder.

Sp. 190-194 ° C.

d) Cis-2,2-dimethyl-5- [N- (p-tolylsulfonyl) -20-amino] -6- [N- (p-tolylsulfonyl) -N- (Ν'-2-p-tolylsulfonylaminomethyl) ] - 1,3-dioxepane disodium salt 87.8 g of the substance prepared according to Example 5c are slurried in 410 ml of ethanol, the mixture is heated to boiling and a solution of 6.67 g of sodium in 200 ml of ethanol is added dropwise. The solution is cooled in an ice bath, 450 ml of ether are added and the precipitate formed is filtered off with suction and dried at 80 ° C and 200 torr to give 91 g of the title compound as a white powder.

30 e) 4,5-Bis- (hydroxymethyl) -3,6,9-tritosylsulfonyl-3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13- triene acetonide 78.75 g of the disodium salt of Example 5d are dissolved in 880 ml of dimethylformamide, the solution is heated to 35-100 ° C and a solution of

II

33 94758 19.53 g of bis- (2,6-chloromethyl) pyridine in 360 ml of dimethylformamide and the solution is heated at 120 DEG C. for a further 5 hours, after which it is evaporated in vacuo to 300 ml. The solution is stirred in 5 liters of ice water, the precipitate formed is filtered off with suction, washed with water and dried. The crude product is crystallized from 700 ml of dioxane to give 45 g of the title compound as a white powder.

Sp. 244-250 ° C.

f) 4,5-Bis- (hydroxymethyl) -3,6,9,15-tetraaza-bicyclo [9.3.1] pentadeca-1 (15), 11,13-triene acetonide in 260 ml of liquid ammonia a slurry of 20 g of the substance obtained in Example 5e in 140 ml of tetrahydrofuran is added, the mixture is stirred in a cold bath at -50 [deg.] C. and a total of 14.4 g of sodium is added in small portions. The mixture is stirred for a further 5 hours at -60 [deg.] C., after which the cold bath is removed and 50 ml of ethanol are added dropwise to the mixture and the ammonia is allowed to evaporate, then the mixture is further evaporated to dryness in vacuo and the residue is chromatographed on silica gel. Elute with chloroform / ethanol / concentrated ammonia (3: 1: 0.5) to give 5.30 g of the title compound as an oil.

Analysis: C 62.72; H 8.55; N 18.29 (calc.) 25 C 62.51; H 8.41; N 18.45 (tod.).

g) 4,5-Bis- (hydroxymethyl) -3,6,9,15-tetraaza-bicyclo [9.3.1] pentadeca-1 (15), 1,13-triene-3,6,9-tris - (acetic acid tert-butyl ester) acetonide

To a solution of 4 g of the substance prepared according to Example 1f in 100 ml of tetrahydrofuran and 10 ml of water are added 5.51 g of anhydrous sodium carbonate and 10.2 g of bromoacetic acid tert-butyl ester and stirred for 5 hours at 50 ° C. :in. The mixture is filtered and evaporated to dryness in vacuo, after which the oily residue is taken up in 50 ml of hexane and decanted.

The residue is purified by chromatography on 100 g of silica gel eluting with dichloromethane (1-10% ethanol) to give 5.7 g of the title compound as a bright yellow oil.

5 h) 4,5-Bis- (hydroxymethyl) -3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9- tris-acetic acid

A mixture of 5.3 g of the ester prepared according to Example Ig and 50 ml of trifluoroacetic acid is stirred for 10 hours at 50 ° C. 10 ml of water are then added to the mixture, it is further stirred for 2 hours at 50 [deg.] C. and then evaporated to dryness in vacuo. The residue is dissolved in 20 ml of water and the solution is loaded onto a column containing. ® ... packed in 100 ml of Reillex (= poly-4-vinylpyridine), eluted with 100 ml of water and the eluate is evaporated to dryness in vacuo. An amorphous powder is obtained which still contains 8.5% of water. The yield is 2.90 g.

Analysis: C 51.81; H 6.41; N 12.72 (calc.) 20 C 51.63; H 6.70; N 12.51 (tod.).

i) 4,5-Bis- (hydroxymethyl) -3,6,9,15-tetraaza-bicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris -acetic acid gadolinium complex

A mixture of 2 g (water content 8.5%, corresponding to * 2.83 g = 4.29 mmol) of 4,5-bis- (hydroxymethyl) -3,6,9,12-tetraazabicyclo [9.3. 1] pentadeca-1 (15), 11,13-triene-3,6,9-tris-acetic acid (preparation: see Example 5h) and 778 mg of gadolinium oxide are stirred for 5 hours at 90 ° C in 50 ml of with water. After cooling, the mixture is stirred alternately with 10 ml of anion exchanger IRA-410 and cation exchanger IRC-50, filtered and lyophilized. This gives 2.35 g of the title compound as a fluffy white powder with a water content of 7.3% by Karl-Fischer titration.

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Analysis (corrected for water content) C18 H 2: C 38.38; H 4.24; N 9.42; Gd 26.44 (calc.) C 38.51; H 4.31; N 9.36; Gd 26.19 (tod.).

Example 6 a) 4,5-Bis- (hydroxymethyl) -3,6,9-tris- [2-dihydro- (3H) -furanon-3-yl] -3,6,9,15-tetra -azabicyclo- [9.3.1] pentadeca-1 (15), 11,13-triene acetonide

To a solution of 5 g (16.34 mmol) of 4,5-bis- (hydroxymethyl) -3,6,9,12-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11, 13-triene acetonide in 100 ml of acetonitrile, 12 g of potassium carbonate, 260 mg of potassium iodide and 13.50 g of α-bromo-7-butyrolactone are added and the mixture is boiled for 48 hours. The mixture is then evaporated to dryness in vacuo, the residue is dissolved in methylene chloride and the mixture is shaken several times with water, the organic phase is dried over sodium sulfate and evaporated to dryness in vacuo. The oily residue is chromatographed on 150 g of silica gel eluting with methylene chloride / methanol 20 (15: 1) to give 5.3 g of the title compound as a bright yellow viscous oil.

b) 4,5-Bis- (hydroxymethyl) -3,6,9,15-tetraaza-bicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris - [α- (2-hydroxyethyl)] - acetic acid 25 g of the compound prepared according to Example 6a are dissolved in 50 ml of water and the pH of the solution is adjusted to 2 by adding hydrochloric acid. The solution is refluxed for 5 hours, then cooled to room temperature and loaded onto a column equipped with ...

30 g of 10 g of Reillex preparation (= poly-4-vinylpyridine), which is then washed with 20 ml of water and from which the eluates obtained and combined are freeze-dried. This gives 4.05 g of the title compound as a fluffy powder with a water content of 7.2%.

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Analysis (corrected for water content) for C 25 H 40 N 4 OH: C, 52.44; H 7.04; N 9.78 (calc.) C 52.61; H 7.33; N 9.62 (sec.) · 5 c) 4,5-Bis- (hydroxymethyl) -3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11.13 -triene-3,6,9-tris- [α- (2-hydroxyethyl)] - acetic acid gadolinium complex

A mixture of 1.50 g (2.68 mxnol) of 4,5-bis- (hydroxymethyl) -3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-10 1 (15), 11, 13-Triene-3,6,9-tris- [α- (2-hydroxyethyl)] acetic acid and 487 mg of gadolinium oxide in 25 ml of water are stirred for 4 hours at 90 ° C. After cooling, the solution is mixed in turn with 5 ml of anion exchanger IRA-410 and 5 ml of cation exchanger IRC-50, the solution is filtered and lyophilized to give 1.69 g of the title compound as a white powder with a water content of 4.3 %.

Analysis (corrected for water content) for C 25 H 37 GdN 4 Ou: 20 C 41.31; H 5.13; N 7.71; Gd 21.63 (calc.) C 41.07; H 5.33; N 7.61; Gd 21.89 (tod.).

Example 7 a) 4-Hydroxymethyl-3,6,9-tritosyl-3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene 25 To a solution of 59 .57 g of 3-aza-1-hydroxymethyl-1,3,5-tritosylpentanediamine (preparation: see

WO patent application PCT / DE 88/00200, WO 88/08422, page 45) In 500 ml of dimethylformamide, 9.60 g of a 50% slurry of sodium hydride in mineral oil are added in small portions, after which the solution is heated for 80 hours. At * ° C. To this solution is added dropwise a solution of 17.61 g of 2,6-bis- (chloromethyl) pyridine in 150 ml of dimethylformamide, followed by heating at 110 ° C for 6 hours. The mixture is evaporated in vacuo to about 220 ml and 1 liter of water is added dropwise, the precipitate formed is filtered off with suction, washed with water and dried overnight at 50 ° C and 200 mbar. The crude product is recrystallized from 500 ml of ethanol to give 45 g of the title compound as a yellow solid.

b) 4-Hydroxymethyl-3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene 42 g of the compound obtained according to Example 7a are heated in 120 ml of concentrated sulfuric acid 48 for 100 hours at 10 ° C. Cool to 0 ° C and add dropwise 350 ml of diethyl ether. This precipitates the salt of the title compound. It is filtered off with suction and dissolved in 100 ml of water, then 40 g of sodium hydroxide are added to the solution and the mixture is extracted several times with dichloromethane. The combined organic phases are dried over sodium sulphate and then evaporated to dryness in vacuo to give 13.3 g of the title compound as a viscous oil.

c) 4-Hydroxymethyl-3,6,9,15-tetraazabicyclo- [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-triacetic acid 20 10 g of the compound prepared according to Example 7b the amine is dissolved in 100 ml of water and 13.21 g of chloroacetic acid are added to the solution. The mixture is stirred at 60 ° C for 5 hours, during which time the pH is maintained at 9.0 by the addition of 10 N sodium hydroxide solution. The mixture is cooled to 0 [deg.] C., 100 ml of ethanol are added to it and it is acidified to pH 1 by adding concentrated hydrochloric acid, the precipitate formed is filtered off with suction, dissolved in 50 ml of water and the solution is loaded onto a 30 ml ® column. ...

Reillex (= poly-4-vinylpyridine), which is then washed with 50 ml of water, and the eluates obtained are combined and lyophilized. This gives 16.8 g of the title compound as an amorphous powder with a water content of 9.3%.

38 94758

Analysis (corrected for water content) for C18H26N4Ov: C, 52.68; H 6.39; N 13.65 (calc.) C 52.49; H 6.54; N 13.81 (tod.).

5 d) Gadolinium complex of 4-hydroxymethyl-3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-triacetic acid

To a solution of 2.3 g of 4-hydroxymethyl-3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11.13-10 triene in 30 ml of water is added 1.015 g gadolinium oxide and the mixture is heated at 90 ° C for 1 hour. A small amount of unreacted oxide is filtered off, after which the solution is passed alternately through 10 ml of anion exchanger IRA-410 and a cation exchanger IRC-50, the ion exchanger is washed with 30 ml of water and the combined eluates are freeze-dried. This gives 3.05 g of the title compound as a powder with a water content of 7.5%.

Analysis (corrected for water content): C 38.29; H 4.11; N 9.92; Gd 27.85 (calc.) 20 C 38.44; H 4.32; N 9.68; Gd 27.71 (tod.).

Example 8 a) 4-Benzyloxymethyl-3,6,9,15-tetraazabicyclo [9.3. 1] pentadeca-1 (15), 11,13-triene-3,6,9-triacetic acid To a solution of 10 g (= 24.36 mmol) of 4-hydroxymethyl-3,6,9, 15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-triacetic acid (prepared according to Example 7b) in 150 ml of dimethylformaroid, 0.5 g of potassium iodide, 4 , 17 g of benzyl bromide and 5 g of sodium carbonate. The mixture is heated at 60 [deg.] C. for 20 hours, then evaporated to dryness in vacuo and 100 ml of water and 300 ml of ethanol are added to the residue and the pH is adjusted to 2 by adding concentrated hydrochloric acid. The precipitate formed is filtered off with suction, dissolved in 35 to 100 ml of water and the solution is applied to a column of 39 94758 ® ......

50 g of Reillex (= poly-4-vinylpyridine), which are then washed with 50 ml of water and the resulting combined aqueous phases are freeze-dried. This gives 8 g of the title compound as an amorphous powder.

Analysis: C 59.99; H 6.44; N 11.19 (calc.) C 59.71; H 6.49; N 11.38 (tod.).

b) 4-Benzyloxymethyl-3,6,9,15-tetraazab-10-cyclo [9.3.1] pentadeca-1 (15), 11,13-trylene-3,6,9-triacetic acid gadolinium complex

To a solution of 5 g of 4-benzyloxymethyl-3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-triacetic acid in 100 ml water, 1.81 g of gadolinium oxide are added, the mixture is heated for 3 hours at 80-90 ° C, filtered and the solution is passed alternately through 15 ml of anion exchanger IRA-410 and cation exchanger IRC-50, the columns being washed with 75 ml: 11a of water and the resulting combined aqueous phases are freeze-dried to give 5.85 g of the title compound as an amorphous powder with a water content of 7.4%.

Analysis (corrected for water content): C 45.86; H 4.46; N 8.56; Gd 24.02 (calc.) C 45.69; H 4.71; N 8.72; Gd 23.81 (tod.).

Example 9 3,6,9,15-Tetraazabicyclo [9.3. 1] pentadeca-3,6,9-tris- [α- (hydroxymethyl) acetic acid] gadolinium complex 4.5 g (7.2 mmol) of the title compound of Example 2c are dissolved in 150 ml of deionized water and the solution is hydrogenated in an autoclave using rhodium catalyst (5% Rh / C) at 30 bar and 40 ° C. After 12 hours, the catalyst is removed by filtration and the filtrate is stirred alternately with 3 ml of cation exchanger (IR 120) and 3 ml of 35 anion exchangers (IRA 410) for 1 hour. The ion exchanger is filtered off and the solution is lyophilized.

40 9 4 7 5 8

Yield: 4.18 g (92% of theory) of a colorless, amorphous powder (containing 6.7% of water by analysis).

Analysis (corrected for water content): δ C 30.08; H 5.27; N 8.88; Gd 24.93 (calc.) C 30.01; H 5.34; N 8.78; Gd 24.86 (tod.) ·

Example 10

Preparation of a solution of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3,6,9-tris- [α- (2-10 hydroxyethyl) acetic acid ] from the gadolinium (3) complex a) 361 g (0.5 mol) of the complex obtained according to Example Ib (water content: 8.3%) are dissolved in 500 ml of water for injection (pi) by gentle heating. 0.8 g of tromethamine is added to the solution, after which the volume of the solution is made up to 1000 ml by adding p.i.

The solution is ultrafiltered and filled into flasks. After thermal sterilization, the contents of the vials are ready for use outside the gastrointestinal tract for diagnostics.

e • i

IJ

Claims (5)

1. Macrocyclic compounds having a 3,6,9,15-tetra-azabicyclo [9.3.1] pentadecane structure and having the general formula (I) g / Cx X1 T X1 il in TOOC-CH M N-CH-COOZ / τ \ B1 C1 CH-X2 where C00Z ... represents a single bond or a double bond, Q represents a nitrogen atom or group NH, X1 and X2 independently represent a hydrogen atom or a group "(CH2) n-OH or - (CH 2) m - (CH) n-CH 2 OH, in which n is one of the numbers 1-5 and OH m is one of the numbers 0-2, A1, B1, C 'and D1 independently of each other a hydrogen atom or a group of benzyloxymethyl or - (CH 2) n-OH, where n is one of the numbers 1 to 5, G represents a hydrogen atom or another macrocyclic group of general formula (II) I [II '(JI) ZOOC-CH N N-CH-COOZ 35 - CH-X 2 COZ 44 94758 and Z represents a hydrogen atom and / or a metal ion equivalent of an element of the order numbers 21 - 29, 31, 32, 37 - 39, 42 - 44, 49 or 57 - 83, 5 provided that X1 and X2 are only in the fa In both hydrogen atoms, when at least one of the four ring substituents A1, B1, C1 and D1 is other than a hydrogen atom. Use of at least one physiologically acceptable compound according to claim 1 for the preparation of a composition used for NMR, X-ray or radio diagnostics. 3. Compositions used in diagnostics, characterized in that they contain at least one compound according to claim 1, possibly together with conventional galenic additives. 4. Compositions used in diagnostics, characterized in that they contain at least one compound of claim 1 in the form of liposomes. 20 Process for the preparation of compositions according to claim 3, characterized in that an aqueous or a physiological saline solution dissolved or suspended, possibly together with conventional galenic additives, is brought into a form suitable for enteral or parenteral administration. 1 9. . A