DE3713842A1 - SUBSTITUTED CYCLIC COMPLEX MAKERS, COMPLEX AND COMPLEX SALTS, METHOD FOR THE PRODUCTION THEREOF AND PHARMACEUTICAL AGENTS CONTAINING THEM - Google Patents

Abstract

Compounds of general formula (I), where q stands for the numbers 0, 1, 2 or 3, A stands for the group (a), in which m = 1, 2, 3, 4 or 5, l = 0, 1, 2, 3, 4 or 5, R<1> is a straight-chain, branched, saturated or unsaturated C0-C20 alkylene group which may contain imino, phenylenoxy, phenylenimino, amido, hydrazido, ester groups, oxygen, sulphur and/or nitrogen atoms, possibly substituted by hydroxy, mercapto, imino, epoxy, oxo, thioxo and/or amino groups, having either a terminal ring of general formula (II), where A<1> stands for the (b) group, a terminal functional group or a bio- or macromolecule bound to this functional group, B, D, and E, which are the same or different, each stand for the (c) group where R<2> is hydrogen or R<1>, k = 0, 1, 2, 3, 4 or 5, n = 0 or 1, Z stands for hydrogen or CH2X, where X stands for the residues -COOY or -PO3HY where Y is a hydrogen atom and/or a metallic ion equivalent of one of the ordinal numbers 21-29, 31, 32, 37-39, 42-44 or 57-83, provided that B, E, and D each contain not less than 2 and not more than 5 carbon atoms and that at least two Zs stand for CH2X, as well as their salts with inorganic and/or organic bases or amino acids, are useful diagnostic and therapeutic agents.

Description

The invention relates to the subject matter characterized in the claims, that is, new complexing agents, complexes and complex salts, these compounds containing agents, their use in diagnostics and therapy, and methods for the preparation of these compounds and agents.

The use of complexing agents or complexes or their salts in medicine has long been known. Examples include:
Complexing agents as stabilizers of pharmaceutical preparations, complexes and their salts as auxiliaries for the administration of poorly soluble ions (e.g. iron), complexing agents and complexes (preferably calcium or zinc), optionally as salts with inorganic and / or organic bases, as Antidots for detoxification in the event of inadvertent incorporation of heavy metals or their radioactive isotopes and complexing agents as aids in nuclear medicine using radioactive isotopes such as ^99m TC for scintigraphy are known.
In the patent DE-OS 34 01 052, paramagnetic complex salts have recently been proposed as diagnostics, predominantly as NMR diagnostics.

All previously known complexes and their salts prepare in their clinical Application Compatibility and / or selectivity problems the bond and / or stability. These problems are all the more pronounced, ever higher the products derived from the complexing agents must be dosed. The use of heavy elements as components of X-ray contrast media to be administered parenterally have so far failed due to the insufficient tolerance of such compounds. So far for the Magnetic resonance imaging proposed or tested paramagnetic substances is the distance between the effective and the toxic in animal experiments Dose relatively narrow, and / or they have a low organ specificity and / or Stability and / or contrast-enhancing effect on and / or their tolerance ness is insufficient.  

The approach to at least part of these problems by using complex formers, on the one hand, by ionic bonding to the appropriate metal (see below) and on the other hand by binding to a functional group or a non-toxic and possibly organ-specific serving as carrier molecule fish macromolecule bound to solve was previously only very limited successful.

Become the functional groups of the complexing agent for binding the molecule used on a macromolecule, the complex is weakened stability, that is, a physiologically intolerable proportion of the metal ions of the macromolecule-metal ion complex is released [C.H. Paik et al., J. Radioanal. Chem. 57, 553 (1980), D.J. Hnatowich et al., J. Nucl. Med. 26, 503 (1985)].

On the other hand, if bifunctional complexing agents are used, that is Complexing agents that contain both functional groups for coordinative binding of the desired metal ion as well as a (other) functional group for binding of the macromolecule, according to the current state of the art (C.F. Meases et al., Radioimmunoimaging and Radioimmunotherapy 1983, 185, Canadian Patent No. 11 78 951) the various serious disadvantages; to the Example low stability of the complexes, multi-stage difficult synthesis of the Complex, low variation possibilities for binding to the macromolecule required functional group, risk of contamination of the complexing agents during their synthesis with foreign metal, due to insufficient lipophilicity only limited reaction possibilities of the complexing agents, with reduction in yield and necessary intermediate blockade associated with additional cleaning steps the functional groups of the complexing agents (for example as an iron complex or protection of a phenolic hydroxy group as methyl ether), need work with highly purified solvents and equipment.

There is therefore a need for stable, readily soluble, and sufficiently selective, but also better tolerated, easily accessible Complex compounds that have the greatest possible variety for binding Have macromolecules of suitable functional groups. The invention lies hence the task of making these connections and means available provide, as well as the simplest possible process for their preparation create. This object is achieved by the invention.  

It has been found that compounds derived from the anion of a functional based complex-forming cyclic carbon or phosphonic acid and one or several central ions of an element of atomic numbers 21-29, 31, 32, 38, 39, 42-44 or 57-83 and optionally one or more cations of an anor ganic and / or organic base or amino acid, surprisingly excellent for the production of NMR, X-ray and radio diagnostics as well as radiotherapeutics.

The compounds of the invention are represented by the general formula I described

wherein

q for the digits 0, 1 or 2, for the group in which
m the digits 1, 2, 3, 4 or 5,
l the digits 0, 1, 2, 3, 4 or 5,
R ^{1 '} optionally containing an imino, phenyleneoxy, amide, ester group (s), oxygen, sulfur and / or nitrogen atom (s), if appropriate by hydroxyl, mercapto, imino and / or amino group (n) Substituted straight-chain, branched, saturated or unsaturated C₁-C₂₀ alkylene group, which in the end either a ring of the general formula II where A¹ for the group stands,
has a functional group or a macromolecule bonded via this functional group,
means
B, E and D, which are the same or different, each for the group With
R ^{2 '} in the meaning of hydrogen or R ^1' ,
k has the meaning of the digits 0, 1, 2, 3, 4 or 5,
n in the meaning of the digits 0 or 1,
X for the radicals -COOY or -PO₃HY with Y in the meaning of a hydrogen atom and / or a metal ion equivalent of an element of atomic numbers 21-29, 31, 32, 38, 39, 42-44 or 57-83
stand,
with the proviso that B, E and D each contain at least 2 and at most 5 carbon atoms,

and their salts with inorganic and / or organic bases or amino acids.

Compounds of general formula I with Y in the meaning of hydrogen are used as complexing agents and with at least two of the substituents Y in the Meaning of a metal ion equivalent referred to as metal complexes.

The element of the above atomic number, which is the central ion of the physio logical compatible complex salt forms, can be aimed for Of course, the intended use of the diagnostic agent according to the invention be radioactive.

If the agent according to the invention is intended for use in NMR diagnostics, the central ion of the complex salt must be paramagnetic. These are esp especially the divalent and trivalent ions of the elements of atomic numbers 21-29, 42, 44 and 58-70. Suitable ions are, for example, chromium (III), manganese (II), Iron (II) -, cobalt (II) -, nickel (II) -, copper (II) -, praseodymium (III) -, Neodymium (III), samarium (III) and ytterbium (III) ion. Because of their very strong magnetic moments are particularly preferred the gadolinium (III) -, terbium (III) -, Dysprosium (III), holmium (III), erbium (III) and iron (III) ion.  

For the use of the agents according to the invention in nuclear medicine Diagnostics, the central ion must be radioactive. For example, are suitable Radioisotopes of the elements copper, cobalt, gallium, germanium, yttrium, strontium, Technetium, indium, ytterbium, gadolinium, samarium and iridium.

If the agent according to the invention is intended for use in X-ray diagnostics, so the central ion must be derived from an element of higher atomic numbers, to achieve adequate absorption of the X-rays. It was found that for this purpose diagnostic agents that are a physiological compatible complex salt with central ions of elements of atomic numbers contained between 21-29, 42, 44, 57-83, are suitable; these are for example the lanthanum (III) ion and the above-mentioned ions of the lanthanide series.

The alkylene group contained in R ^{1 '} can be straight-chain, branched, cyclic, aliphatic, aromatic or arylaliphatic and have up to 20 carbon atoms. Straight-chain mono- to hexamethylene groups and C₁-C₄-alkylenephenyl groups are preferred. If the alkylene group contains a phenoxy group, this is preferably bonded p-permanently to the -CH group of the basic structure of the compound of the general formula I via a methylene group.

Preferred functional groups which are located at the end of the R ^{1 '} alkylene group are, for example, the benzyl ester, ethyl ester, t-butyl ester, amino, C₁-C₆ alkylamino, aminocarbonyl, hydrazino, hydrazinocarbonyl, maleimido -, Methacrylamido, methacryloyl hydrazinocarbonyl, maleimidamidocarbonyl, halogeno, mercapto, hydrazinotrimethylene hydrazinocarbonyl, aminodimethylene amocarbonyl, bromocarbonyl, phenylenediazonium, isothiocyanate, semicarbazide, thiosemicarbazide group.

A few selected R ^{1 ′} substituents are listed for clarification:

Contain the compounds according to the invention (one) ring (s) of the general Formula II, the link is preferably made via the groups

If not all acidic hydrogen atoms are substituted by the central ion , one, more or all of the remaining hydrogen atoms (e) can be Cations of inorganic and / or organic bases or amino acids can be replaced. Suitable inorganic cations are, for example, lithium ion, potassium ion, the calcium ion and especially the sodium ion. Suitable organic cations Bases include those of primary, secondary or tertiary Amines, such as ethanolamine, diethanolamine, morpholine, glucamine, N, N-dimethylglucamine and especially N-methylglucamine. Suitable cations of Amino acids are, for example, those of lysine, arginine and ornithine.

The complexing agents according to the invention (Y = hydrogen) are produced in that in a manner known per se in compounds of the general Formula III

wherein

A'for the group

B ', E' and D ', which are the same or different, each for the group where R ¹ 'optionally containing an imino, phenyleneoxy, phenyleniomino, amide, ester group (s), oxygen, sulfur and / or nitrogen atom (s), optionally by hydroxy, mercapto, imino and / or amino group (s) substituted straight-chain, branched, saturated or unsaturated C₁-C₂₀-alkyl group, which in the end either a ring or the general formula II ' where R ² ′ is hydrogen or R ¹ ′
or mean a functional group
X ′ represents the radicals -COOY ′ or PO₃Y ′ with Y ′ in the meaning of an acid protecting group,
the protective groups Y 'are split off and the acids of the formula III' thus obtained with Y 'in the meaning of a hydrogen atom, if desired
a) in a manner known per se with at least one metal oxide or metal salt of an element of atomic numbers 21-29, 31, 32, 38, 39, 42-44, 49 or 57-83 and then, if desired, acidic hydrogen atoms present by cations inorganic and / or organic bases or amino acids substituted, or
b) in a manner known per se with at least one metal oxide or metal salt of an element of atomic numbers 21-29, 31, 32, 38, 39, 42-44, 49 or 57-83 and then reacting the metal complexes thus obtained in a manner known per se binds to a macromolecule via the functional group contained in R ¹ 'and, if desired, substitutes existing acidic hydrogen atoms by cations of inorganic and / or organic bases or amino acids, or
c) binds to a macromolecule in a manner known per se via the functional group contained in R ¹ ′ and then in a manner known per se with at least one metal oxide or metal salt of an element of atomic numbers 21-29, 31, 32, 38, 39, 42-44, 49 or 57-83 and then, if desired, existing acidic hydrogen atoms substituted by cations of inorganic and / or organic bases or amino acids.

The acid protecting groups Y 'are lower alkyl, aryl and aralkyl groups, for example the methyl, ethyl, propyl, butyl, phenyl, benzyl, diphenyl methyl, triphenylmethyl, bis (p-nitrophenyl) methyl group, as well as trialkylsilyl groups in question.
The protecting groups Y 'are split off by the processes known to those skilled in the art, for example by hydrolysis, alkaline saponification of the esters with alkali in aqueous alcoholic solution at temperatures from 0 to 50 ° C. or in the case of tert-butyl esters with the aid of trifluoroacetic acid.

The starting materials are prepared by cyclization of two reactants, one of which is R ^{1 ′ ′} and the other R ^{2 ′ ′ is} substituted, where R ^{1 ′ ′ is} a substituent which can be converted into R ^{1 ′} and R ^{2 ′ '} Represent hydrogen or R ^1' ; the cyclic compounds obtained in this way are then, if appropriate after splitting off N-protecting groups, with an ester of the general formula IV

HalCH₂X ′ (IV)

wherein
Hal stands for chlorine, bromine or iodine, and
X 'has the meaning given for the general formula III,

alkylated.

The cyclization is carried out according to methods known from the literature (for example Org. Synth. 58, 86 (1978), Makrocyclic Polyether Syntheses, Springer Verlag Berlin, Heidelberg, New York 1982, Coord. Chem. Rev. 3, 3 (1968), Ann. Chem. 1976, 916): one of the two reactants carries two leaving groups at the end of the chain, the other two nitrogen atoms, which nucleophilically displace these leaving groups. Examples include the reaction of terminal diazaalkylene compounds, optionally containing one or two nitrogen atoms, dibromo, dimesyloxy, ditosyloxy or dialkoxycarbonylalkylene compounds, optionally containing one or two additional nitrogen atoms in the alkylene chain, of which one of the two reactants R ^{1 ''} -, the other R ^{2 '' is} substituted.
The nitrogen atoms are optionally protected, for example as tosylates, and are released before the subsequent alkylation reaction by methods known from the literature.
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.

The subsequent alkylation with halogen esters of the general formula IV takes place in polar aprotic solvents such as dimethylformamide, Dimethyl sulfoxide or hexamethylphosphoric triamide in the presence of an acid scavengers such as tertiary amine (e.g. triethylamine, trimethyl amine, N, N-dimethylaminopyridine, 1,5 diazabicyclo [4.3.0] nonen-5 [DBN], 1.5 diazabicyclo [5.4.0] undecene-5), alkali, alkaline earth carbonate or bicarbonate (for example sodium, magnesium, calcium, barium, potassium carbonate and -hydrogen carbonate) at temperatures between -10 ° C and 120 ° C, preferably between 0 ° C and 50 ° C.

Compounds with more than one ring are synthesized by methods known from the literature, for example via an addition / elimination reaction of an amine of a carbonyl compound (for example acid chloride, mixed anhydride, activated ester, aldehyde); two amine-substituted rings with a dicarbonyl compound (for example oxalyl chloride, glutardialdehyde); two rings, each having a nucleophilic group, with an alkylene compound bearing two leaving groups or, in the case of terminal acetyls, by oxidative coupling (Cadiot, Chodkiewicz in Viehe "Acetylenes", 597-647, Marcel Dekker, New York, 1969).
The chain linking the rings can then be modified by subsequent reactions (for example hydrogenation).

Suitable substituents R ^{1 ''} are, inter alia, hydroxy and nitrobenzyl, hydroxy and carboxyalkyl and thioalkyl radicals having up to 10 carbon atoms. They are converted into the desired substituents (for example with the amino-) using the literature methods known to those skilled in the art (Chem. Pharm. Bull. 33, 674 (1985), Compendium of Org. Synthesis Vol. 1-5, Wiley and Sons, Inc.). , Hydrazino-, Hydra zinocarbonyl-, Methacryloylhoydrazinocarbonyl-, Maleimidamidocarbonyl-, Halogeno-, Halogenocarbonyl-, Mercapto group as functional group) converted, whereby in the case of the nitrobenzyl residue first a catalytic hydrogenation (for example according to PN Rylander, Catalytic Hydrogenation over Platinum Metals, Academ Press 1967) for the aminobenzyl derivative.
Examples of the conversion of hydroxyl or amino groups bound to aromatic or aliphatic radicals are those in anhydrous, aprotic solvents such as tetrahydrofuran, dimethoxyethane or dimethyl sulfoxide in the presence of an acid scavenger such as sodium hydroxide, sodium hydride or alkali metal or alkaline earth metal carbonates such as sodium, Magnesium, potassium calcium carbonate at temperatures between 0 ° C and the boiling point of the respective solvent, but preferably between 20 ° C and 60 ° C, carried out reactions with a substrate of the general formula V.

Z-L-Fu (V)

wherein

For a nucleofuge such as e.g. B. Cl, Br, J, CH₃C₆H₄SO₃ or CF₃SO₃, L for an aliphatic, aromatic, arylaliphatic, branched, straight chain or cyclic hydrocarbon radical with up to 20 carbon atoms and Stand for the desired terminal functional group.

Examples of compounds of the general formula V are mentioned

Conversions of carboxy groups can, for example, according to the carbodiimide Method (Fieser, Reagents for Organic Syntheses 10, 142) on a mixed Anhydride [Org. Prep. Proc. Int. 7, 215 (1975)] or via an activated ester (Adv. Org. Chem. Part B, 472).

The preparation of those required as starting substances for the cyclization Amines are carried out analogously to methods known from the literature.  

Starting from an N-protected amino acid, one obtains by reaction with a partially protected diamine (for example using the carbodiimide method), Deprotection and diborane reduction a triamine.

The reaction of a diamine obtainable from amino acids (Eur. J. Med. Chem.-Chim. Ther. 21, 333 (1986)) with twice the molar amount of an N-protected ω- amino acid provides a tetramine after suitable work-up.

In both cases the number of carbon atoms is between the N atoms by the type of diamines or amino acids used as coupling partners determinable.

The complex-forming acids obtained in this way can also be linked to macromolecules which are known to accumulate particularly well in the organ or organ part to be examined. Such macromolecules are, for example, enzymes. Hormones, sugars, dextrans, lectins, prophyrins, bleomycins, insulin, prostaglandins, steroid hormones, amino sugars, amino acids, peptides such as polylsin, proteins (such as immunoglobulins and monoclonal antibodies) or lipids (also in the form of liposomes). Of particular note are conjugates with albumins such as human serum albumin, antibodies such as monoclonal antibodies specific for tumor associated antigens, antimyosin or cholic acid. Instead of biomolecules, suitable synthetic polymers such as polyethylenimines can also be attached. The pharmaceutical agents formed therefrom are suitable, for example, for use in tumor and infarct diagnostics and tumor therapy. As monoclonal antibodies (for example Nature 256, 495, 1975), which have the advantages over the polyclonal antibodies that they are specific for an antigenic determinant, have a defined binding affinity, are homogeneous (this makes their purification much easier) and that can be produced in large quantities in cell cultures, in particular those for the conjugation are those which are directed against predominantly cell membrane permanent antigens. As such, for example, for tumor imaging, monoclonal antibodies or their fragments Fab and F (ab ′) ₂ are suitable, which are, for example, specific for human tumors of the gastrointestinal tract, breast, liver, bladder, gonads and Melanomas (Cancer Treatment Repts. 68, 317, 1984, Bio Sci 34, 150, 1984) or against carcinoembryonic antigen (CEA), human choriogonadotropin ( β- HCG) or other tumorigenic antigens such as glycoproteins. (New Engl. J. Med. 298, 1384, 1973, U.S. Patent 4,331,647). Anti-myosin, anti-insulin and anti-fibrin antibodies (US Pat. No. 4,036,945) are also suitable.

Are suitable for liver examinations or for tumor diagnosis for example conjugates or inclusion compounds with liposomes (the for example as unilamellar or multilamellar phosphatidylcholine cholesterol Vesicles are used).

The bindings known from the prior art, for example of radioiso tops to immunoglobulins and their fragments, have the disadvantage of insufficient stability of the labeled antibody conjugates or insufficient specificity (for example as a result of the use of a diethylenetriaminepentaacetic acid = DTPA anhydride) (for example Diagnostic Imaging 84, 56; Science 220, 613, 1983; Cancer Delivery 1, 125, 1984).
The conjugate formation according to the present invention, on the other hand, takes place via the functional group located at the end of the C₁-C₂₀alkylene group of the substituent R¹, as defined above. When the complex-forming acids are conjugated with proteins, peptides or lipids, several acid residues can be bound to the macromolecular biomolecule. In this case, each complex-forming acid residue can carry a central ion.

The coupling to the desired macromolecules is also per se known methods, as described, for example, in Rev. Roum. Morphole. Embryo. Physio., Physiologie 1981, 18, 241 and J. Pharm. Sci. 68, 79 (1979) are, for example by reaction of the nucleophilic group of a macromolecule, such as the amino, phenol, sulfhydryl, aldehyde or imidazole group an activated derivative of the complexing agent. Come as capitalized derivatives for example monoanhydrides, acid chlorides, acid hydrazides, mixed anhydrides (See, for example, G.E. Krejcarek and K.L. Tucker, Biochem., Biophys. Res. Commun. 1977, 581), activated esters, nitrenes or isothiocyanates. Conversely, it is also possible to use an activated macromolecule with the complex bil implement the acid. Substi are also available for conjugation with proteins tuenten for example of the structure C₆H₄N₂, C₆H₄NHCOCH₂, C₆H₄NHCS or C₆H₄OCH₂CO at.  

In the case of the antibody conjugates, the binding of the antibody to the Complexing agent (or to the metal complex; the production of the metal complex Conjugate can be in the order of complexing agent, complexing agent conjugate, End product as well as in the order complexing agent, metal complex, end product) not to lose or reduce the binding affinity and binding specificity of the antibody to the antigen. This can either by binding to the carbohydrate portion in the Fc portion of the glycoprotein or in the Fab or F (ab ′) ₂ fragments or by binding to sulfur atoms of the anti body or the antibody fragments.

In the first case, an oxidative cleavage of sugar units must first Generation of formyl groups capable of coupling. This oxidation can be chemically treated with oxidizing agents such as periodic acid, Sodium metaperiodate or potassium metaperiodate according to methods known from the literature (for example J. Histochem. and Cytochem. 22, 1084, 1974) in aqueous solution in Concentrations of 1 to 100, preferably 1 to 20 mg / ml, and a concentration tration of the oxidizing agent between 0.001 to 10 mmol, preferably 1 to 10 mmol in a pH range of approx. 4 to 8 at a temperature between 0 to 37 ° C and a reaction time between 15 minutes and 24 hours will. The oxidation can also be carried out enzymatically, for example with With the help of galactose oxidase in an enzyme concentration of 10-100 units / ml, a substrate concentration of 1 to 20 mg / ml, at a pH of 5 to 8, a reaction time of 1 to 8 hours and a temperature between 20 and 40 ° C (for example J. Biol. Chem. 234, 445, 1959).

The aldehydes generated by oxidation become complexing agents (or metal complex, see above) with suitable functional groups such as Hydrazine, hyrazide, primary amine, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide by reaction between 0-37 ° C, with a reaction time from 1 to 65 hours, a pH between about 5.5 and 8, an antibody concentration of 0.5 to 20 mg / ml and a molar ratio of the complex bildners for antibody aldehyde from 1: 1 to 1000: 1 bound. The subsequent one The conjugate is stabilized by reducing the double bond, e.g. B. with sodium borohydride or sodium cyanoborohydride; the reducing agent will used in a 10 to 100-fold excess (for example J. Biol. Chem. 254, 4359, 1979).  

The second possibility for the formation of antibody conjugates starts from one gentle reduction of the disulfide bridges of the immunoglobulin molecule; here the more sensitive disulfide bridges of the H chains of the antibody Molecule cleaved while the S-S bonds of the antigen-binding region are intact remain so that there is practically no reduction in binding affinity and -specificity of the antibody occurs (Biochem. 18, 2226, 1979, Handbook of Experimental Immunology, Vol. 1, Second Edition, Blackwell Scientific Publications. London 1973, Chapter 10). These free sulfhydryl groups of the intra-H chains Regions are then labeled with suitable functional groups of complexing agents or metal complexes at 0 to 37 ° C, a pH of about 4 to 7, and one Reaction time of 3 to 72 hours with formation of a covalent bond, which does not affect the antigen binding region of the antibody. As Suitable reactive groups may be mentioned, for example: haloalkyl, Haloacetyl, p-mercuribenzoate groups and groups which have a Michael addition Reaction such as maleimides, methacrylic groups (e.g. J. Amer. Chem. Soc. 101, 3097, 1979).

Conditions of a non-convex nature can also be used for coupling, with ionic as well as van der Waals and hydrogen bonds in varying proportions and strength (key-lock principle) for binding can contribute (for example avidin-biotin, antibody-antigen). Also a final connections (host-guest) of smaller complexes in larger cavities at the macro molecules are possible.

A method which is particularly suitable for producing conjugates of both antibody and antibody fragments is coupling to a solid phase. Here, the antibody or the corresponding F (ab) ₂ fragment is bound to a stationary phase (for example an ion exchanger), which is located in a temperature-controlled column with inflow and outflow. To oxidize the Fc part of the antibody, the column must be protected from light by covering; To reduce disulfide bridges (for example when generating Fab fragments) it must be possible to work under argon as a protective gas. The actual coupling process then proceeds as follows:
After rinsing the column with a suitable buffer, a solution is used as the eluent, which generates reactive groups on the bound protein (for example, periodate solution to generate aldehyde groups in the Fc part of monoclonal antibodies or mercaptoethylamine solution to produce sulfhydryl groups in Fragments). After the reaction solution has completely displaced the previous eluent, the flow is stopped for a time sufficient for complete reaction, then rinsed sufficiently with buffer, then a solution is drawn with the coupling partner (for example the hydrazide or dithiopyridyl derivative of a complexing agent or a complex) opens and stops the flow again for a sufficiently long time. Instead of stopping the flow for a long time, a so-called recycle circuit can also be used; the eluate leaving the column is pumped directly back onto the column by means of a loop circuit. One achieves much shorter reaction times and better yields because of the better mixing. This is followed by a rinse with buffer solution. If a free complexing agent is the coupling partner, complexing is carried out in a further cycle with a solution of the desired metal salt (for example a citrate solution) and a subsequent rinsing cycle. Finally, the conjugate is eluted with a pH or salt gradient. Then, if necessary after desalting, lyophilization. After equilibration with buffer solution, the column is ready for the next coupling run.
This method is far superior both in speed and in yield to the preparation of very small and very large amounts of conjugate, and also allows the continuous production of conjugates; this is the prerequisite for the economical production of large quantities.

The compounds formed in this way are then preferably purified chromatographically via ion exchangers on a fast protein liquid chromatography system.
The compounds of the general formula I thus obtained with Y in the meaning of a hydrogen atom represent complexing agents. They can be isolated and purified or converted into metal complexes of the general formula I with at least two of the substituents Y in the meaning of a metal ion equivalent without isolation.

The metal complexes according to the invention are prepared in the manner as it is disclosed in the patent specification DE-OS 34 01 052 by the Metal oxide or a metal salt (for example the nitrate, acetate, carbonate, Chloride or sulfate) of the element of atomic numbers 21-29, 31, 32, 38, 39, 42-44, 49, 57-83 in water and / or a lower alcohol (such as methanol), ethanol or isopropanol) dissolves or suspended and with the solution or suspension of equivalent amount of the complexing acid of the general formula I with Y in the meaning of a hydrogen atom and then, if desired, existing acidic hydrogen atoms of acid groups by cations of inorganic and / or organic bases or amino acids.

The neutralization takes place with the help of inorganic bases (for example Hydroxides, carbonates or bicarbonates) of, for example, sodium, potassium or Lithium and / or organic bases such as primary, secondary and tertiary amines such as ethanolamine, morpholine, glucamine, N-methyl and N, N-dimethylglucamine, and basic amino acids, such as lysine, Arginine and ornithine.

To produce the neutral complex compounds, for example, the acidic complex salts in aqueous solution or suspension as much of the desired Add bases so that the neutral point is reached. The solution obtained can then concentrated to dryness in vacuo. It is often an advantage the neutral salts formed by adding water-miscible solution agents such as lower alcohols (methanol, ethanol, isopropanol and others), lower ketones (acetone and others), polar ethers (tetrahydrofuran, Dioxane, 1,2-dimethoxyethane and others) precipitate and so easily isolating and easy to clean crystals. As special It has proven advantageous to have the desired base already during the Add complex formation to the reaction mixture and thereby a process save step.

If the acidic complex compounds contain several free acidic groups, it is often useful to produce neutral mixed salts that are both inorganic and also contain organic cations as counterions.  

This can be done, for example, by adding the complexing acid to aqueous suspension or solution containing the oxide or salt of the central ion Elements and half of the amount required for neutralization converts organic base, the complex salt formed is isolated, if desired cleans and then for complete neutralization with the required amount inorganic base added. The order of adding bases can also be reversed will.

The conjugates of antibody and complex are used before in vivo use Incubation with a weak complexing agent, such as sodium citrate, Sodium ethylenediaminetetraacetic acid dialysed to weakly bound metal to remove atoms.

In the case of using radioisotopes containing complex compounds their manufacture according to the "Radiotracers for Medical Applications", Volume 1, CRC-Press, Boca Raton, Florida will.

The pharmaceutical compositions according to the invention are also produced in a manner known per se by using the complex compounds according to the invention - optionally with the addition of the additives customary in galenics - in aqueous Medium suspended or dissolved and then the suspension or solution sterilized if necessary. Suitable additives are, for example, physiological harmless buffers (such as tromethamine), small additions of Complexing agents (such as diethylenetriaminepentaacetic acid) or, if required, electrolytes such as sodium chloride or, if required Lich, antioxidants such as ascorbic acid.

Are suspensions or for enteral administration or other purposes Solutions of the agents according to the invention in water or physiological saline they are desired with one or more auxiliaries customary in galenics substance (s) (for example methyl cellulose, lactose, mannitol) and / or surfactant (s) (for example lecithins, Tween®, Myrj® and / or flavoring (s) for Flavor correction (for example essential oils) mixed.  

In principle, it is also possible to use the pharmaceutical compositions according to the invention can also be produced without isolating the complex salts. In any case, must be special Care should be taken to chelate so that the Salts and salt solutions according to the invention are practically free of not complex ionic metal ions.

This can be done, for example, with the help of color indicators such as xylenol orange Control titrations can be guaranteed during the manufacturing process. The The invention therefore also relates to processes for the preparation of the complex compounds and their salts. As a final security, cleaning of the isolated remains Complex salt.

Are suspensions of the complex for oral administration or other purposes compounds in water or physiological saline is desired, a sparingly soluble complex compound with one or more commonly used in galenics Auxiliary (s) and / or surfactant (s) mixed. To correct the taste Flavors are added.

The pharmaceutical compositions according to the invention preferably contain 1 µmol-1 mol / l of the complex salt and are usually in amounts of 0.001-5 mmol / kg dosed. They are intended for enteral and parenteral applications.

The complex compounds according to the invention are used

• 1. for NMR and X-ray diagnostics in the form of their complexes with the ions of Elements with atomic numbers 21-29, 42, 44 and 57-83;
• 2. for radio diagnostics and radiotherapy in the form of their complexes with the Radioisotopes of elements with atomic numbers 27, 29, 31, 32, 38, 39, 43, 49, 64, 70 and 77.

The agents according to the invention meet the diverse requirements for Suitability as a contrast medium for magnetic resonance imaging. So they are forth excellently suitable, after oral or parenteral application by elevation the signal intensity in the image obtained with the aid of an MRI scanner to improve its informative value. Furthermore, they show the high effectiveness that is necessary to the body with the smallest possible amounts of foreign substances strain and the good tolerance that is necessary to the non-invasive To maintain the character of the investigations.

The good water solubility of the agents according to the invention makes it highly concentrated Manufactured solutions so that the volume load of the circuit in keep reasonable limits and dilution by body fluid to compensate, that means NMR diagnostics have to water 100-1000 times better be soluble than for NMR spectroscopy. Furthermore, the fiction not only have high stability in vitro, but also Surprisingly high stability in vivo, so that a release or an exchange of the ions which are not covalently bound in the complexes - which are toxic in themselves - within half the time in which the new contrast agents are completely excreted are done extremely slowly.

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

The agents according to the invention are due to their favorable radioactive properties and the good stability of the complex compounds contained in them also suitable as radiodaignostika. Details of their application and dosage z. B. "Radiotracer for Medical Applications", CRC-Press, Boca Raton, Florida described.

Another imaging method with radioisotopes is the positron Emission tomography, the positron emitting isotopes such. B. ⁴³Sc, ⁴⁴Sc, ⁵²Fe, ⁵⁵Co and ⁶⁸Ga used. (Heiss, W.D., Phelps, M.E., Position Emission Tomography of Brain, Springer Verlag Berlin, Heidelberg, New York 1983).  

The compounds of the invention can also be used in radioimmunotherapy be applied. This only differs from the corresponding diagnostics by the amount and type of radioactive isotope used. The goal is there the destruction of tumor cells by high-energy short-wave radiation the shortest possible range. The specificity of the antibody used is of crucial importance here as non-specifically localized antibodies conjugate leads to the destruction of healthy tissue.

The antibody or the antibody fragment of the antibody-metal complex according to the invention serves to transport the complex to the target organ in an immunospecific manner for the antigen in question, where the metal ion selected for its cell-killing properties can emit rays which lethally damage the cells. Suitable b -emitting ions are, for example, ⁴⁶Sc, ⁴⁷Sc, ⁴⁸Sc, ⁷²Ga and ⁷³Ga. Suitable low half-life α- emitting ions are, for example, ²¹¹Bi, ²¹²Bi, ²¹³Bi and ²¹⁴Bi, with ²¹²Bi being preferred.

In the in vivo application of the therapeutic agents according to the invention can be combined with a suitable carrier such as serum or physiological saline and together with another protein such as Example human serum albumin can be administered. The dosage is included depending on the type of cellular disorder, the metal ion used and the type of imaging method.

The therapeutic agents according to the invention are preferred parenterally i.V. applied.

The agents according to the invention are outstandingly suitable as X-ray contrast agents, It should be emphasized that they do not show any signs of of the anaphylaxis-like reactions known from the iodine-containing contrast media biochemical-pharmacological investigations. Particularly worth it they are full due to the favorable absorption properties in higher areas Tube voltages for digital subtraction techniques.  

In general, the agents according to the invention are for use as X-rays Contrast medium in analogy to, for example, meglumine diatrizoate in amounts of 0.1-5 mmol / kg, preferably 0.25-1 mmol / kg.

Details of the use of X-ray contrast media are, for example, in Barke, X-ray contrast media, G. Thieme, Leipzig (1970) and P. Thurn, E. Bücheler - "Introduction to X-Ray Diagnostics", G. Thieme, Stuttgart, New York (1977) discussed.

Overall, new complexing agents, metal complexes and metal have been successful to synthesize complex salts, the new possibilities in diagnostic and open up therapeutic medicine. Above all, the development of a new kind of image Giving procedures in medical diagnostics leaves this development appear desirable.

The following examples serve to explain the counterpart of the invention in more detail befitting.  

Example 1 a) O-Benzyl-N-trifluoroacetyltyrosine

11.5 g (0.41 mmol) of O-benzyltyrosine are suspended in 1 l of dry methanol and 58.9 ml (0.42 mol) of triethylamine are added at room temperature. After the addition of 67 ml (0.53 mol) of trifluoroacetic acid ethyl ester is stirred for 130 hours at room temperature with the exclusion of water. It is separated from unreacted starting material and volatile components are removed by shaking with ethyl acetate / aqueous hydrochloric acid. The ethyl acetate phase is decolorized with activated carbon. After evaporation of the solvents, 120.7 g (80% of theory) of colorless crystals are obtained.
Melting point: 149-150 ° C

Analysis:

b) O-Benzyl-N-trifluoroacetyltyrosine (2-carbobenzoxyaminoethylene) amide

18.5 g (50.4 mmol) of O-benzyl-N-trifluoroacetyltyrosine (Example 1a) are dissolved in 200 ml of dry tetrahydrofuran, mixed with 7 ml of Et₃N and then added dropwise 4.8 ml (50.8 mmol) of ethyl chloroformate, keeping the temperature below -10 ° C. After the addition has ended, the mixture is stirred at this temperature for 30 minutes, the same amount of precooled triethylamine is added, and an ice-cold solution of 11.6 g (50.4 mmol) of N- (2-aminooethyl) -carbamic acid benzyl ester hydrochloride in 100 ml of dimethylformamide dripped. The mixture is stirred for a further 30 minutes at -10 ° C., then allowed to come to room temperature with stirring and then warmed to 30 ° C. for 10 minutes. The solvent is then removed on a rotary evaporator and poured onto 750 ml of ice water. The crystals are filtered off, washed with ice water and dried. The yield is 26.9 g (94% of theory)
Melting point: 189-190 ° C

Analysis:

c) O-benzyltyrosine (2-carbobenzoxyaminoethylene) amide

25.9 g (47.8 mmol) of O-benzyl-N-trifluoroacetyltyrosine (2-carbobenzoxyamino ethylene) -amide (Example 1b) are suspended in 300 ml EtOH and portions 7.2 g (191 mmol) of sodium borohydride were added. After stirring overnight at room temperature, 50 ml of acetone are added and several times with vinegar ester extracted. The organic phase provides after drying and concentration 18.8 g (88% of theory) of white crystals with a melting point of 145 ° C.

Analysis:

Calcd .: C 69.77 H 6.53 N 9.38 O 14.29 Found: C 69.79 H 6.53 N 9.35

d) tyrosine (2-aminoethylene) amide

42.3 g (96.4 mmol) of O-benzyltyrosine (2-carbobenzoxyaminoethylene) amide (Example 1c) are dissolved in 1.1 l of methanol, 2 g of 10% palladium-carbon are added and the mixture is hydrogenated with stirring until none Hydrogen uptake occurs more. The catalyst is filtered off and the solvent is evaporated off. It is dissolved in methanol in the heat and precipitated with ether: 17 g (86% of theory) of colorless crystals.
Melting point: 138-141 ° C

Analysis:

Calc .: C 59.17 H 7.67 N 18.81 O 14.33 Found: C 59.23 H 7.51 N 18.90

e) 3-aza-1- (4-hydroxybenzyl) pentane-1,5-diamine · trihydrochloride

6.55 g (29.3 mmol) of tyrosine (2-aminoethylene) amide (Example 1d) are suspended in 130 ml of dry tetrahydrofuran and a slow stream of B₂H₆ (from 5.8 g of NaBH₄ in 75 ml of diethylene glycol dimethyl ether and 54 ml Boron trifluoride etherate complex) with dry nitrogen with constant stirring through the solution. The mixture is stirred at 60 ° C. overnight, then 30 ml of methanol are added dropwise at 20 ° C. and hydrogen chloride is passed in with ice cooling. Then briefly boil up and suck off. The trihydrochloride is obtained in the form of colorless crystals (8.04 g; 86% of theory).
Melting point: 250 ° C (decomposition)

Analysis:

f) 3-Aza-1- (4-hydroxybenzyl) -1,3,5-tri-tosyl-pentane-1,5-diamine

24.0 g (75.3 mmol) of 3-aza-1- (4-hydroxybenzyl) pentane-1,5-diamine (as trihydrochloride) are placed in 250 ml of dry pyridine and 37 at 0 ° C. with good stirring , 9 g (198.7 mmol) of tosyl chloride, dissolved in 100 ml of pyridine, were added dropwise over an hour. The mixture is left to stand at 4 ° C. overnight, the main part of the pyridine is evaporated off in vacuo and taken up in 300 ml of dichloromethane. By repeated washing with dilute hydrochloric acid, aqueous bicarbonate solution and bidistilled water, residual pyridine and excess toluene sulfonic acid are removed. After drying, it is chromatographed on silica gel with ethyl acetate-hexane. The tritosylate is obtained in the form of colorless crystals. Yield 36.9 g (73% of theory).
Melting point: 145-146 ° C

Analysis:

g) 2- (4-Hydroxybenzyl) -1,4,7,10-tetra-tosyl-1,4,7,10-tetraazacyclododec-an

10.95 g (16.3 mmol) 3-aza-1- (4-hydroxybenzyl) -1,3,5-tri-tosyl-pentane-1,5- diamine are dissolved in 100 ml dimethylformamide and with 1.35 g (45 mmol) Sodium hydride (80% in paraffin) stirred at 35-40 ° C for 30 minutes. After that ver is slowly added with a solution of 9.51 g (16.3 mmol) of 3-aza-1,3,5- tritosyl-1,5-dihydroxypentane in 100 ml DMF. The mixture is stirred for four hours at 130 ° C. allows to cool overnight and the solvent is distilled off. The residue crystallizes when triturated with a little methanol. After washing with ver thinner hydrochloric acid and recrystallization from acetonitrile gives 7.4 g (48% of theory) colorless crystals with a melting point of 192-193 ° C.

Analysis:

h) 2- (4-hydroxybenzyl) -1,4,7,10-tetraazacyclododecane · trihydrobromide

12.0 g (12.7 mmol) of 2- (4-hydroxybenzyl-1,4,7,10-tetra-tosyl-1,4,7,10-tetra Azacyclododecane in 50 ml glacial acetic acid with 33% hydrogen bromide for four hours heated to 100 ° C. It is poured into 300 ml of diethyl ether, suction filtered and resuspended twice in 300 ml of diethyl ether each. All operations will be carried out under a protective nitrogen atmosphere. After drying in a vacuum there are 5.16 g (78% of theory) of colorless crystals at 115-117 ° C. melt with decomposition.

Analysis:

i) 2- (4-hydroxybenzyl) -1,4,7,10-tetrakis (tert-butoxy-carbonylmethyl) - 1,4,7,10- tetraazacyclododecane

8.49 g (16.3 mmol) of 2- (4-hydroxybenzyl) -1,4,7,10-tetraazacyclododecane suspended in 150 ml of dimethylformamide with 9.66 g (115 mmol) of sodium hydro Gen carbonate added and at 60 ° C with a solution of 12.7 g (65.2 mmol) Tert-butyl bromoacetate in 100 ml of DMF. After 2 hours Stirring at this temperature, the solvent is removed and the oily Chromatograph the residue on silica gel with ether / hexane. You get one colorless viscous oil. Yield 9.0 g (79%).

Analysis:

Calc: C 63.73 H 9.05 N 7.62 O 19.59 Found: C 63.70 H 8.97 N 7.50

j) 2- (4-hydroxybenzyl) 1,4,7,10-tetrakis (carboxymethyl) -1,4,7,10-tetraazacy clododecane

1.66 g (2.38 mmol) of 2- (4-hydroxybenzyl) -1,4,7,10-tetrakis (tert-butoxycarbonyl methyl) -1,4,7,10-tetraazacyclododecane are dissolved in 50 ml of trifluoroacetic acid Stirred at 35 ° C for 2 hours. Then poured into 250 ml of dry ether, suction filtered, slurried with ether several times and dried for 24 hours at 50 ° C in a vacuum. After recrystallization from dichloromethane / tetrahydrofuran, 1.13 g (93%) of colorless crystals are obtained.
Melting point: 238 ° C (decomposition)

Analysis:

Calc: C 54.10 H 6.71 N 10.97 O 28.20 Found: C 54.17 H 6.52 N 10.93  

Gadolinium complex

6.28 mg (12.3 mmol) of the complexing agent is dissolved in 20 ml of 0.1 N ammonium acetate. Solution and mixed with 12.5 ml of a 0.9857 n gadolinium acetate solution. Man increases the pH to 7.5 with dilute ammonia solution and heats for 30 minutes to 80 ° C. After centrifugation, the supernatant is freeze-dried. You get 8.1 g of the complex (99%) as a white powder.

Analysis:

Sodium salt of the Gd complex

3.78 g (5.69 mmol) of the complex are dissolved in 25 ml of water, with 10.6 ml a 1.073 n sodium hydroxide solution was added and freeze-dried. 4.03 g remain (quantitative) of a white powder.

Analysis:

N-methylglucamine salt of the Gd complex

2.0 g (3.0 mmol) of the complex are combined with 1.17 g (6.0 mmol) of N-methyl glucamine, dissolved in 100 ml of water, filtered and freeze-dried. It stays 3.1 g (98%) of colorless crystals.

Analysis:

Instead of gadolinium acetate, other salts of gadolinium or gadolinium oxide can also be used if the respective counterions form volatile ammonium compounds.
By using appropriate salts or oxides of other metals such as Mu (II); Co (III); Cu (II); Dy; La; Y; Sm; Ho; Yb; Bi; Fe (III) or Pb gives their complexes and complex salts.

Instead of 3-aza-1- (4-hydroxybenzyl) -1,3,5-tritoxylpentan-1,5-diamine (Example 1f), the corresponding 2- (4-hydroxybenzyl) isomer can also be used which can be manufactured in the following way:

α ) 3-Aza-2- (4-benzyloxybenzyl) -4-oxoglutaric acid diamide (Method A)

3.62 g (13.3 mmol) of O-benzyltyrosinamide are refluxed with 2.7 g of ethyl oxamate (23 mmol) in dimethoxyethane for 14 hours. After the solution has been stripped off, it is washed successively with water, ethanol and ether. After drying, 2.73 g of white crystals (60% of theory) are obtained.
Melting point: 270 ° C

Analysis:

Calc: C 63.33 H 5.61 N 12.30 O 18.74 Found: C 63.24 H 5.52 N 12.14

or according to method B

• α 1) 3-aza-2- (4-benzyloxybenzyl) -4-oxoglutaric acid 5-ethyl ester-1-amide;
  3 g (11.1 mmol) of O-benzyltyrosinamide are dissolved in 30 ml of dimethoxyethane, 1.56 ml of triethylamine are added and 1.53 g (11.1 mmol) of oxalic acid ethyl ester chloride are added dropwise at 0.degree. After 30 minutes at 0 ° C., the mixture is poured onto 100 ml of ice, filtered off with suction and dried. The yield is 3.87 g (94% of theory).
  Melting point: 142 ° C Analysis: Calc.: C 64.85 H 5.98 N 7.56 O 21.59 Found: C 64.71 H 6.11 N 7.46
• α 2) 3.6 (9.72 mmol) 3-aza-2- (4-benzyloxybenzyl) -1-oxoglutaric acid-5-ethyl ester-1-amide (Example α 1) are mixed with 40 ml of a solution of 1 mol NH₃ / l poured methanol. After 1 hour, the precipitated product is filtered off. After drying, 3.13 g (95% of theory) of the title compound are obtained in the form of colorless crystals.
  Melting point: 269 ° C Analysis: Calc.: C 63.33 H 5.61 N 12.30 O 18.74 Found: C 63.25 H 5.63 N 12.17

β ) 3-Aza-2- (4-hydroxybenzyl) -4-oxoglutaric acid diamide

1 g (2.9 mmol) of 3-aza-2- (4-benzyloxybenzyl) -4-oxoglutaric acid diamide (Example α ) is suspended with 100 mg of 10% palladium-carbon and a few drops of concentrated hydrochloric acid in 20 ml of methanol and to the end the hydrogen uptake hydrogenated. After filtering off the catalyst, 690 mg of colorless crystals (93% of theory) are obtained.
Melting point: 245-250 ° C (decomposition)

Analysis:

Calc: C 52.58 H 5.21 N 16.72 O 25.47 Found: C 52.83 H 5.19 N 16.84

γ ) 3-aza-2- (4-hydroxybenzyl) pentane-1,5-diamine · trihydrochloride

1 g (4.0 mmol) of 3-aza-2- (4-hydroxybenzyl) -4-oxoglutaric acid diamide (Example β ) are reacted according to the instructions given for Example 1e. The colorless crystals obtained weigh 1.19 g (93.7% of theory).
Melting point: 238 ° C

Analysis:

δ ) 3-Aza-2- (4-hydroxybenzyl) -1,3,5-tri-tosylpentane-1,5-diamine

Analogously to the procedure given for 1f, 3.64 g (80% of theory) of the title compound are obtained starting from 2.16 g of the starting material prepared according to γ ).
Melting point: 175-177 ° C

Analysis:

Example 2 a) 2- [4- (3-Benzyloxycarbonylaminopropoxy) benzyl] -1,4,7,10-tetrakis (te-rt.- butoxycarbonylmethyl) -1,4,7,10-tetraazacyclododecane

9.5 g (13.6 mmol) of 2- (4-hydroxybenzyl) -1,4,7,10-tetrakis (tert-butoxy carbonylmethyl) -1,4,7,10-tetraazacyclododecane (Example 1i) with 261 mg Sodium hydride (80% in paraffin oil, 13.6 mmol) dissolved in 100 ml of toluene and slowly at 30 ° C with a solution of 3.84 g (14.9 mmol) of N- (3-bromopropyl) - benzyl carbamate in 20 ml of toluene. After 2 hours one turns on concentrated and freed of paraffin via a silica gel. 11.15 g are obtained a colorless oil (88.5% of theory).

Analysis:

Calc: C 64.83 H 8.59 N 7.56 O 19.0 Found: C 64.81 H 8.65 N 7.60

b) 2- [4- (3-aminopropoxy) benzyl] -1,4,7,10-tetrakis (tert-butoxycarbonyl-methyl) - 1,4,7,10-tetraazacyclododecane

10.52 g (11.36 mmol) of 2- [4- (3-benzyloxycarbonylaminopropoxy) benzyl] -1,4,7,10- tetraktis (tert-butoxycarbonylmethyl) -1,4,7,10-tetraazacyclododecane are hydrogenated in 55 ml of methanol with 1 g of 10% palladium-carbon until none further hydrogen uptake occurs. After filtering off the catalyst and concentration, 8.55 g (95% of theory) of a colorless oil remain.

Analysis:

Calc: C 63.86 H 9.28 N 8.84 O 18.17 Found: C 63.50 H 9.33 N 8.72

c) 2- [4- (3-aminopropoxy) benzyl] -1,4,7,10-tetrakis (carboxymethyl) -1,4, -7.10- tetraazacyclododecane

According to regulation 1j, the tert-butyl esters can be cleaved with trifluoroacetic acid from 2- [4- (3-aminopropoxy) benzyl] -1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) -1,4,7 , 10-tetraazacyclododecane in 86% yield.
Melting point: 195 ° C (decomposition)

Analysis:

Calc: C 55.01 H 7.28 N 12.33 O 25.36 Found: C 55.20 H 7.31 N 12.33

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

Gd complex Na salt of the Gd complex N-methylqlucamine salt of the Gd complex Example 3 a) 2- [4-3- (Maleimido) propoxybenzyl] -1,4,7,10-tetrakis (tert-butoxycarbonyl) methyl) -1,4,7,10-tetraazacyclododecane

7.92 g (10 mmol) of 2- [4- (3-aminopropoxy) benzyl] -1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) -1,4,7,10-tetraazacyclododecane (example 2b) in 200 ml of dichloromethane, 980 mg (10 mmol) of maleic anhydride are stirred overnight at 40 ° C. and then 1.35 g (10 mmol) of N-hydroxybenzotriazole and 2.26 g (11 mmol) of dicyclohexylcarbodiimide are added at room temperature. After 2 days, the mixture is filtered and the product is purified by chromatography (dichloromethane / ether; silica gel). A colorless oil is obtained.
Yield: 6.63 g (73% of theory)

Analysis:

Calc: C 63.35 H 8.43 N 8.03 O 20.18 Found: C 63.27 H 8.40 N 8.13

b) 2- [4-3- (Maleimido) propoxybenzyl] -1,4,7,10-tetrakis (carboxymethyl) - 1,4,7,10- tetraazacyclododecane

6.6 g (7.56 mmol) of 2- [4-3- (maleimido) propoxybenzyl] -1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) -1,4,7,10-tetraazacyclododecane are dissolved in 50 ml of trifluoroacetic acid and stirred at 30 ° C for 3 hours. It is poured into 10 times the amount of dry diethyl ether, suction filtered and dried in vacuo. 4.33 g (88.5% of theory) of colorless crystals are obtained.
Melting point: above 175 ° C (decomposition)

Analysis:

Calc: C 55.63 H 6.38 N 10.81 O 27.17 Found: C 55.63 H 6.25 N 10.72  

Gd complex

8.16 g (12.6 mmol) of 2- [4-3- (maleimido) propoxybenzyl] -1,4,7,10-tetrakis (carboxymethyl) - 1,4,7,10-tetraazacyclododecane are in 100 ml of 0.1 N ammonace Tat solution dissolved and with 12.8 ml of a 0.9857 n-cadolinium acetate solution transferred. The pH is adjusted to 7.5 with dilute ammonia solution, heated 20 minutes at 80 ° C and centrifuged. After freeze-drying, the supernatant delivers 10.0 g (99% of theory) of colorless crystals.

Analysis:

Sodium salt of the Gd complex

2.16 g (2.69 mmol) of the complex are dissolved in 20 ml of water and 2.5 ml a 1.073 n sodium hydroxide solution. After freeze drying, 1.77 g (99% of theory).

Analysis:

N-methylglucamine salt of the Gd complex

3.25 g (4.05 mmol) of the complex, dissolved in 50 ml of water, is mixed with 791 mg (4.05 mmol) of N-methylglucamine were added in portions at 40-45 ° C. After more complete Dissolution of the base is freeze-dried. 4.05 g (quant.) Of colorless are obtained Crystals.

Analysis:

Example 4 a) 2- [4- (Oxiranylmethoxy) benzyl] -1,4,7,10-tetrakis (tert-butoxycarbon-yl methyl) -1,4,7,10-tetraazacyclododecane

12.72 g (18.2 mmol) of 2- (4-hydroxybenzyl) -1,4,7,10-tetrakis (tert-butoxycar bonylmethyl) -1,4,7,10-tetraazacyclododecane (Example 1i) dissolved in 250 ml of toluene and mixed with 350 mg of sodium hydride (80% in paraffin, 18.3 mmol). The mixture is heated to 80 to 100 ° C. and a solution of 1.74 g (18.2 mmol) of epichlorohydrin in 50 ml of toluene is added dropwise. After heating for two hours at the reflux temperature, the mixture is concentrated and purified on a silica gel column. A colorless oil is obtained.
Yield: 11.53 g (79.7% of theory)

Analysis:

Calc: C 63.44 H 9.38 N 7.04 O 20.12 Found: C 63.34 H 9.15 N 7.12

b) 2- [4- (Oxiranylmethoxy) benzyl] -1,4,7,10-tetrakis (carboxymethyl) -1,4-, 7,10- tetraazacyclododecane

As described in Example 1j, 2- [4- (oxiranylmethoxy) benzyl] -1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) -1 is obtained from 5.0 g (6.3 mmol) , 4,7,10-tetraazacyclododecane 3.5 g (97% of theory) of the free acid as colorless crystals.
Melting point: above 160 ° C (decomposition)

Analysis:

Calc: C 55.11 H 6.75 N 9.88 O 28.23 Found: C 55.17 H 6.76 N 9.72  

Gd complex

As described for Example 1j, the Gd complex is obtained quantitatively.

Analysis:

The sodium salt and the N-methylglucamine salt of the complex are also obtained.

Sodium salt of the Gd complex

Analysis:

N-methylglucamine salt of the Gd complex

Analysis:

Example 5 a) 2- [4- 4-aza-2-hydroxy-14- (2-tert-butoxycarbonyl) hydrazinocarbonyl) - tetradecyloxy-benzyl] -1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) -1,4,7,10- tetraazacyclododecane

11.7 g (14.7 mmol) of the compound according to Example 4a are dissolved in 150 ml of diethyl ether and with 4.63 g (14.8 mmol) of 11-aminoundecanoic acid 2- (tert-butoxycarbonyl) hydrazide in 100 ml of tetrahydrofuran added dropwise. The mixture is refluxed for 2 hours, the solvent is removed by distillation and chromatographed. A colorless oil is obtained.
Yield: 13.4 g (81% of theory)

Analysis:

b) 2- [4- 4-aza-2-hydroxy-14-hydrazinocarbonyl-tetradecyloxy-benzyl] -1,4,7,10-- tetrakis (carboxymethyl) -1,4,7,10-tetraazacyclododecane

According to the procedure described for Example 1j, the tert-butyl groups can be split off with trifluoroacetic acid. From 2.66 g (2.36 mmol) of the compound Example 5a, 1.59 g (88% of theory) of the title compounds are thus obtained.
Melting point: above 133 ° C

Analysis:

Calc: C 56.83 H 8.12 N 12.53 O 22.5 Found: C 57.77 H 8.21 N 12.63 O 22.41

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

Gd complex

Analysis:

Sodium salt of the Gd complex

Analysis:

N-methylglucamine salt of the Gd complex

Analysis:

Example 6 a) 2- [4- (2-Propynyloxy) benzyl-1,4,7,10-tetrakis (tert-butoxycarbonylm-ethyl) - 1,4,7,10-tetraazacyclododecane

11.4 g (16.3 mmol) of 2- (4-hydroxybenzyl) -1,4,7,10-tetrakis (tert-butoxycarbonyl) - 1,4,7,10-tetraazacyclododecane (Example 1i) dissolved in 250 ml Toluene, with 344 mg sodium hydride (as a suspension in paraffin) (17.9 mmol) and then dropwise at reflux temperature with a solution of 1.93 (16.5 mmol) 3-bromopropine in 50 ml toluene. After Chromato graphically there are 9.58 g (76% of theory) of a colorless oil.

Analysis:

Calc: C 65.25 H 8.86 N 7.24 O 18.62 Found: C 65.19 H 8.81 N 7.20

b) 2- [4- (2-propynyloxy) benzyl] -1,4,7,10-tetrakis (carboxymethyl) -1,4,7-, 10- tetraazacyclododecane

As described for Example 1j, the free acid can be obtained by treating the tert-butyl ester described above with trifluoroacetic acid. The yield is 88.7% with a 10.6 mmol approach.
Melting point: above 176 ° C (decomposition)

Analysis:

Calcd: C 56.92 H 6.61 N 10.21 O 26.24 Found: C 56.90 H 6.70 N 10.14

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

Gd complex

Analysis:

Sodium salt of the Gd complex

Analysis:

N-methylglucamine salt of the Gd complex

Analysis:

Example 7 a) 1,6-bis- {4- [2,5,8,11-tetrakis (tert-butoxycarbonylmethyl) -2,5,8,11-tetraza cyclododecyl] benzyloxy} -2,4-hexadiine

4.9 g (6.35 mmol) of the compound from Example 6a are dissolved in 200 ml of pyridine dissolved and mixed with 1.26 g (12.7 mmol) of copper (I) chloride. The solution will be saturated with oxygen and then stirred for 2 days at room temperature, while maintaining an oxygen atmosphere. To Concentration is filtered through silica gel and then chromatographed. A colorless oil (2.75 g; 56.2% of theory) is obtained.

Analysis:

Calc: C 65.34 H 8.74 N 7.25 O 18.65 Found: C 65.38 H 8.71 N 7.30

b) 1,6-bis- {4- [2,5,8,11-tetrakis (carboxymethyl) -2,5,8,11-tetraazacyclododecyl] - benzyloxy} -2,4-hexadiene

From 2.05 g (1.33 mmol) of the title compound a), 1.34 g (92% of theory) of the free complexing agent is obtained as a colorless crystalline substance by the method described for Example 1j.
Melting point: above 141 ° C (decomposition)

Analysis:
Calc .: C 57.02 H 6.44 N 10.23 O 26.29 Found: C 57.12 H 6.35 N 10.19

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

Gd complex

Analysis:

Sodium salt of the Gd complex

Analysis:

N-methylglucamine salt of the Gd complex

Analysis:

Example 8 a) N-Carbobenzoxyserin- (2-carbobenzoxyaminoethylene) amide

7.34 g (30.7 mmol) of N-carbobenzoxyerine are reacted and worked up according to the instructions given for Example 1b with the corresponding equimolar amounts of ethyl chloroformate, triethylamine and N- (2-aminoethyl) carbamate benzyl ester hydrochloride. 10.33 g (81% of theory) of colorless crystals are obtained.
Melting point: 167 ° C

Analysis:

Calc: C 60.71 H 6.06 N 10.11 O 23.10 Found: C 60.75 H 5.98 N 10.15

b) (2-aminoethyl) serinamide

13.46 g (32.4 mmol) of N-carbobenzoxyserin- (2-carbobenzoxyaminoethylene) -amide are hydrogenated in 200 ml of methanol in the presence of 1.37 g of 10% palladium / carbon until no more hydrogen is taken up. The catalyst is filtered off and all volatile components in the oil pump are removed. A viscous, partially crystalline oil remains.
Yield 4.67 g (98% of theory)

Analysis:

Calc .: C 40.80 H 8.89 N 28.55 O 21.74 Found: C 40.71 H 8.85 N 28.30

c) 1-hydroxymethyl-1,3,5-triazapentane · trihydrochloride

Analogously to the procedure for Example 1e, the title compound is obtained in 67% yield from the amide described above as a white, crystalline powder.
Melting point: 236 ° C (decomposition)

Analysis:

d) 3-aza-1-hydroxymethyl-1,3,5-tris-tosyl-pentane diamine

Analogously to the procedure of Example 1f, the title compound is obtained in 73% yield from 3-aza-1-hydroxymethyl-pentane-1,5-diamine.
Melting point: 138-140 ° C

Analysis:

e) 2-hydroxymethyl-1,4,7,10-tetra-tosyl-1,4,7,10-tetraazacyclododecane

Analogously to the procedure of Example 1g, the title compound is obtained in 38% yield from 3-aza-1-hydroxymethyl-1,3,5-tris-tosylpentane-1,5-diamine and 3-aza-1,3,5-tritosyl Obtained 1,5-dihydroxy-pentane. Colorless crystals.
Melting point: 172 ° C.

Analysis:

f) 2-hydroxymethyl-1,4,7,10-tetraaza-cyclododecane

Analogously to the procedure of Example 1h, the title compound is obtained in 76% yield from 2-hydroxymethyl-1,4,7,10-tetra-toxyl-1,4,7,10-tetraazacyclododecane receive. A finely crystalline powder with a melting point of 97 ° C. is obtained (Decomposition).

Analysis:

Calc: C 53.43 H 10.96 N 27.69 O 7.90 Found: C 53.63 H 10.81 N 27.37

g) 2-hydroxymethyl-1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) -1,4,7-, 10- tetraazacyclododecane

Analogous to the instructions of Example 1i, the title compound is 81% Yield from 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane and bromoacetic acid Obtain tert-butyl ester as a colorless oil.

Analysis:

Calcd .: C 60.15 H 9.48 N 8.50 O 21.85 Found: C 60.37 H 9.36 N 8.36  

h) 2-hydroxymethyl-1,4,7,10-tetrakis (carboxymethyl) -1,4,7,10-tetraazac-yclodo decan

Analogous to the instructions of Example 1j, the title compound is 78% Yield from 2-hydroxymethyl-1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) - 1,4,7,10-tetraazacyclododecane obtained as a white crystalline powder.

Analysis:

Calcd .: C 46.99 H 6.96 N 12.89 O 33.14 Found: C 46.77 H 6.82 N 12.96

The gadolinium complex and its salts are obtained quantitatively as for example 13 described.

Gd complex

Calc: C 34.68 H 4.62 N 9.51 Gd 26.71
Found:

Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 9 a) 2 - [(3-Benzyloxycarbonylaminopropoxy) methyl] -1,4,7,10-tetrakis (tert -.-but oxycarbonylmethyl) -1,4,7,10-tetrazacyclododecane

Analogously to the procedure of Example 2a, the title compound is obtained in 82% yield from 2-hydroxymethyl-1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) - 1,4,7,10-tetraazacyclododecane (Example 8g) obtained. It's about a colorless oil.

Analysis:

Calc: C 62.16 H 8.89 N 8.23 O 20.7 Found: C 62.31 H 8.90 N 8.15

b) 2- (3-aminopropoxymethyl) -1,4,7,10-1,4,7,10-tetrakis (tert.-butoxycar-bonylmethyl) - 1,4,7,10-tetraazacyclododecane

Analogous to the procedure of Example 2b, the title compound is 95% Yield obtained from the title compound 9a. It is a colorless one Oil.

Analysis:

Calc: C 60.39 H 9.71 N 9.78 O 20.11 Found: C 60.31 H 9.66 N 9.90

2- (3-aminopropoxy-methyl) -1,4,7,10-tetrakis (carboxymethyl) -1,4,7,10-- tetraazacyclododecane

Analogously to the procedure of Example 2c, the title compound is obtained in 76% yield from the title compound 9b as a white crystalline powder.
Melting point: 149 ° C (decomposition)

Analysis:

Calcd .: C 48.87 H 7.58 N 14.24 O 29.29 Found: C 48.91 H 7.56 N 14.10

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 10 a) 2- [3-Maleimido) -propoxymethyl] -1,4,7,10-tetrakis (tert-butoxycarbon-ylmethyl) - 1,4,7,10-tetraazacyclododecane

Analogously to the procedure of Example 3a, the title compound is obtained in yield 62% obtained from the title compound of Example 9b as a colorless oil.

Analysis:

Calc: C 60.35 H 8.72 N 8.79 O 22.10 Found: C 60.31 H 8.77 N 8.87

b) 2- [3-Maleimido) propoxymethyl] -1,4,7,10-tetrakis (carboxymethyl) -1,4-, 7,10- tetraazacyclododecane

Analogously to the procedure of Example 3b, the title compound is obtained in 62% yield from the title compound 10a as a colorless powder.
Melting point: 175 ° C (decomposition)

Analysis:

Calc: C 50.43 H 6.52 N 12.25 O 30.79 Found: C 50.44 H 6.70 N 12.33

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 11 a) 2 - [(Oxiranlymethoxy) methyl] -1,4,7,10-tetrakis (tert-butoxybonylmethyl) - 1,4,7,10-tetraazacyclododecane

11.0 g (17.2 mmol) of the title compound from Example 8g are dissolved in 250 ml Toluene dissolved and mixed with 330 mg of sodium hydride (80% in paraffin, 17.2 mmol). The mixture is heated to 80-100 ° C. and a solution is added dropwise of 1.62 g (17.2 mmol) epichlorohydrin in 50 ml toluene. After two hours Heating to reflux temperature is concentrated and over a silica gel column cleaned. A colorless oil is obtained. Yield: 8.9 g (72% of theory)

Analysis:

Calc: C 60.14 H 9.80 N 7.79 O 22.25 Found: C 60.14 H 9.89 N 7.52

b) 2 - [(Oxiranylmethoxy) methyl] -1,4,7,10-tetrakis (carboxymethyl) -1,4,7-, 10- tetraazacyclododecane

As described in Example 1j, 4.34 g (6.1 mmol) of the title compound of 11a gives 2.60 g (87% of theory) of the free acid as colorless crystals.
Melting point: above 177 ° C (decomposition)

Analysis:

Calcd .: C 48.97 H 6.98 N 11.42 O 32.61 Found: C 48.86 H 6.91 N 11.50

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 12 a) [{-Aza-2-hydroxy-14- (2- (tert-butoxycarbonyl) hydrazinocarbonyl) tetra-- decylocy} methyl] -1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) -1,4,7,10- tetraazacyclododecane

10.3 g (14.3 mmol) of the title compound from Example 11a are dissolved in 150 ml of diethyl ether dissolved and with 4.47 g (14.3 mmol) of 11-amino-undecanoic acid 2- (tert.- butoxycarbonyl) hydrazide in 100 ml of tetrahydrofuran added dropwise. The mixture is refluxed for one hour and the solvent is removed by distillation and chromatographed. A colorless oil is obtained. Yield 11.56 g (77% of theory).

Analysis:

Calcd: C 62.06 H 9.18 N 9.21 O 19.54 Found: C 62.21 H 9.21 N 9.14

b) 2 - [{- aza-2-hydroxy-14-hydrazinocarbonyl-tetradecyloxy} methyl] -1,4,7,10- tetrakis (carboxymethyl) -1,4,7,10-tetraazacyclododecane

According to the procedure described for example 1j, the tert.- Split off butyl groups with trifluoroacetic acid. From 2.5 g (2.36 mmol) of Title compound 12a is thus obtained 1.39 g (85% of theory) of the title compound. Melting point: up to 163 ° C (decomposition)

Analysis:

Calcd .: C 52.75 H 8.42 N 13.89 O 24.93 Found: C 53.71 H 8.45 N 14.16

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 13 a) 2 - [(2-propynyloxy) methyl] -1,4,7,10-tetrakis (tert-butoxycarbonylmethyl) - 1,4,7,10-tetraazacyclododecane

10.4 g (16.3 mmol) of the title compound of Example 8g, dissolved in 250 ml Toluene, with 540 mg sodium hydride (as an 80% suspension in paraffin (18 mmol) and then dropwise at reflux temperature with a solution of 2.01 g (16.9 mmol) of 3-bromopropine in 50 ml of toluene. After Chromato graph are 9.86 g (87% of theory) of a colorless oil.

Analysis:

Calc: C 71.77 H 6.88 N 5.97 O 15.36 Found: C 72.89 H 6.92 N 6.03

b) 2 - [(2-propynyloxy) methyl] -1,4,7,10-tetrakis (carboxymethyl) -1,4,7,1-0- tetraazacyclododecane

As described for Example 1j, the free acid can be obtained by treating the tert-butyl ester described above with trifluoroacetic acid. The yield was 80% in a 10 mmolar batch.
Melting point: above 176 ° C (decomposition)

Analysis:

Calc: C 50.84 H 6.82 N 11.85 O 30.47 Found: C 51.18 H 6.74 N 11.97

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 14 a) 1,6-bis - {[2,5,8,11-tetrakis (tert-butoxycarbonylmethyl) -2,5,8,11-tetraaza cyclododecyl] methoxy} -2,4-hexadiine

4.38 g (6.3 mmol) of the title compound from Example 13a are dissolved in 200 ml of Py ridin dissolved and mixed with 1.25 g (12.6 mmol) of copper (I) chloride. The Lö solution is saturated with oxygen and then stirred for 2 days Room temperature, constantly maintaining an oxygen atmosphere becomes. After concentration, filter through silica gel and then chromato graphed. A colorless oil is obtained (2.62 g = 61% of theory).

Analysis:

Calcd: C 62.13 H 9.12 N 8.05 O 20.69 Found: C 63.24 H 9.02 N 7.98

1,6-bis {[2,5,8,11-tetrakis (carboxymethyl) -2,5,8,11-tetraazacyclododecyl] - methoxy} -2,4-hexadiene

From 3.3 g (2.37 mmol) of the title compound of Example 14a, 1.65 g (74% of theory) of the free complexing agent is obtained as a colorless, crystalline substance by the method described for Example 1j.
Melting point: above 140 ° C (decomposition)

Analysis:

Calc .: C 50.94 H 6.62 N 11.88 O30.54 Found: C 50.40 H 6.56 N 12.03

The gadolinium complex and its salts are contained quantitatively as for Example 1j described.

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 15 Conjugate of 2- [3- (maleimido) propoxymethyl] -1,4,7,10-tetrakis (carboxymethyl) - 1,4,7,10-tetraazacyclododecane with F 17754 00070 552 001000280000000200012000285911764300040 0002003713842 00004 17635ab fragments of the monoclonal antibody 7B10D11 a) Production of F (ab ′) ₂ fragments

16 mg (1000 nmol) of the antibody 7B10D11 in 1 ml of a mixture of 0.1 M acetate buffer (pH 4.5) and 0.1 M saline solution and after addition of 0.3 mg pepsin incubated for 20 hours at 37 ° C. After cleaning over Ultragel ACA (LKB) at pH 7.0 and after freeze-drying, 6.3 mg are obtained (63% of theory) of the desired fragments.

b) Production and coupling of Fab fragments

15 mg (150 nmol) of the fragments obtained according to a) are dissolved in 14.5 ml 0.1 M Phosphate buffer (pH 6.0) dissolved and with 0.15 ml of a 0.1 M mercaptoethyl amine solution in 0.1 M phosphate buffer (pH 6.0) with the addition of 15 mmol ethyl lendiamine tetraacetic acid dissolved. After incubating for two hours at 37 ° C separated under argon protection using a Sephadex G 25 column. A determination The sulfhydryl groups result in 328 nmol SH groups in the reaction mixture.

1.23 mg (2.15 µmol) of the complexing agent described in Example 10b dissolved in 10 ml 0.1 M phosphate buffer (pH 6.0). To do this, add the at 4 ° C Solution of the Fab fragment prepared above and left under gentle overnight Shake (at a maximum of 4 ° C) react. Then you elute over one Cation exchanger, dialyzed against 0.1 M ammonium acetate solution and lyophili siert. 14.1 mg of a white powder are obtained.

1 ml ¹¹¹InCl₃ solution (pH 5.5 83 mCi / ml) become a solution of the conjugate placed in 25 ml buffer (20 mmol sodium acetate, 150 mmol sodium chloride) and incubated for 4 hours. Then add another 5 ml of 0.1 M sodium ace solution, dialyzed and lyphylized. 13.82 mg of white powder are obtained with a specific activity of 5 mCi / mg.  

Example 16 ¹¹¹Indium complex of conjugate of 7B10D11 monoclonal antibody with 2- [4- Aza-2-hydroxy-14-hydrazinocarbonyl-tetradecyloxy-benzyl] -1,4,7,10-te-trakis (carboxymethyl) -1,4,7,10-tetraazacyclododecane

30 nmoles of the antibody are released on a macroporous, strongly acidic cation exchanger bound, previously equilibrated with a 0.1 M sodium acetate buffer (pH 5) was burned and protected from light by aluminum foil Pillar. Then rinsed with 0.03 M sodium periodate in acetate buffer, until the periodate appears in the eluate. You stop rinsing for 30 minutes, then washes with acetate buffer and then draws a solution that is 0.03 m on the obi gene hydrazide (Example 5) and 0.1 m of sodium cyanoborohydride. After 2 For hours, non-coupled complexing agent is eluted with acetate buffer; the Conjugate is eluted with a saline gradient. After desalting is done freeze-dried. 4.5 mg of conjugate are obtained, which were described as in Example 15 ben, is converted into the ¹¹¹-indium complex.

Example 17 a) 3- (4-Hydroxybenzyl) -2,4-dioxo-1,5,8,11-tetraazacyclotridecane

27.6 g (0.1 mol) of 4-hydroxybenzylmalonic acid diethyl ester (at Pharm. Fr. 38 (4), 343-52) with 14.6 g (0.1 mol) of triethyletetraamine in 1200 ml Ethanol heated to reflux for 5 days. It is evaporated to dryness in a vacuum and the residue crystallizes several times from toluene. 7 g (22% of the Theory) of a white powder.

Analysis:

Calcd .: C 60.17 H 7.26 N 17.54 Found: C 60.06 H 7.50 N 17.38  

b) 3- (4-hydrobenzyl) -1,5,8,11-tetraazacyclotridecane tetrahydrochloride

32 g (0.1 mol) of the compound obtained under a) are in 350 ml of tetrahy heated to reflux for 5 hours with 1 mol of diborane. Then you drip while cooling 500 ml of methanol, the solution is saturated with hydrogen chloride and heated to reflux for 30 minutes. It is evaporated to dryness and crystallized the residue from ethanol. 26.3 g (60% of theory) of one are obtained white powder.

Analysis:

Calc: C 43.85 H 7.36 N 12.78 Cl 32.36 Found: C 43.67 H 7.66 N 12.53 Cl 32.10

c) 2- (4-hydroxybenzyl) -1,5,8,11-tetrakis (tert-butoxycarbonylmethyl) -1-, 5,8,11- tetraazacyclotridecane

21.9 g (50 mmol) of the compound obtained under b) and 55.0 g (550 mmol) Potassium hydrogen carbonate are dried in 350 ml of dimethylformamide (over Sodium hydride) and dropwise at 35 ° C with a solution of 28.4 g (200 mmol) tert-butyl bromoacetate in 75 ml of dimethylformamide transferred. After stirring for three hours at 35 ° C., the mixture is filtered off with suction and the filtrate concentrated in vacuo. The residue is dissolved in diethyl ether and unumge set bromoacetic acid tert-butyl ester separated over a silica gel. After concentration of the ether solution, 32.5 g (87% of theory) of one are obtained colorless oil.

Analysis:

Calc .: C 64.14 H 9.15 N 7.48 O 19.22 Found: C 64.22 H 9.32 N 7.29

d) 3- [4-Hydroxybenzyl] -1,5,8,11-tetrakis (carboxymethyl) -1,5,8,11-tetra-azacy clotridecan

As described for Example 1j, the free ligand is obtained from the tetraester described above in 88% yield.
Melting point: above 155 ° C (decomposition)

Analysis:

Calc: C 54.95 H 6.91 N 10.68 O 27.44 Found: C 55.61 H 6.85 N 10.59

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 18 a) 3- [4- (3-Benzyloxycarbonylaminopropoxy) benzyl] -1,5,8,11-tetrakis (te-rt.-bu toxycarbonylmethyl) -1,5,8,1-tetraazacyclotridecane

7.49 g (10 mmol) of the compound obtained under 17c) are mixed with 500 mg of Na triumhydride in 80 ml of tetrahydrofuran dropwise with a solution of 3.3 g Benzyl N- (3-bromopropyl) carbamate in 10 ml of tetrahydrofuran. After stirring overnight at room temperature, the mixture is concentrated, the residue in 100 ml of diethyl ether were added and the solution was filtered through activated carbon. After chromatography on silica (system acetic acid / diethyl ether 2: 1), 3.8 g (40% of theory) of a color are obtained after concentration in vacuo loose oil.

Analysis:

Calc: C 65.07 H 8.78 N 7.44 O 18.70 Found: C 65.15 H 8.91 N 7.25

3- [4- (3-Aminoipropoxy) benzyl] -1,5,8,11-tetrakis (tert-butoxycarbonyl-ylme thyl) -1,5,8,11-tetraazacyclotridecane

5.6 g of the compound obtained under a) are dissolved in ethanol with 0.5 g of 10% Palladium carbon is hydrogenated until no more hydrogen absorption can be determined is. After filtering off the catalyst and evaporating off the solvent the substance is analytically pure in quantitative yield as a colorless oil in front.

Analysis:

Calc: C 64.07 H 9.37 N 8.68 O 17.86 Found: C 64.35 H 9.40 N 8.72  

3- [4- (3-aminopropxy) benzyl] -1,5,8,11-tetrakis (carboxymethyl) -1,5,8-, 11- tetraazacyclotridecane

As described for Example 1j, the title compound is obtained from (7.3 mmol) 3- [4- (3-aminopropoxy) benzyl] -1,5,8,11-tetrakis (tert-butoxy carbonylmethyl-) 1 , 5,8,11-tetraazacyclotridecane in 73% yield as a white powder.
Melting point: 192 ° C

Analysis:

Calc: C 54.95 H 6.91 N 10.68 O 27.44 Found: C 54.06 H 6.84 N 10.7

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 19 a) 3- [4- (3-Maleimido-propoxy) benzyl-1,5,8,11-tetrakis (tert-butoxycarbonyl) methyl) -1,5,8,11-tetraazacyclotridecane

Analogously to example 3a), the compound shown under 18a) is obtained tion in 72% yield the title compound as a colorless oil.

Analysis:

Calc: C 63.70 H 8.53 N 7.90 O 19.86 Found: C 64.00 H 8.37 N 7.93

b) 3- [4- (3-Maleimidopropoxy) benzyl] -1,5,8,11-tetrakis (carboxymethyl) - 1,5,8,11- tetraazacyclotridecane

According to the procedure given for Example 3b, the complexing agent is obtained as a white powder from the tetraester prepared under a) in 80% yield
Melting point: above 110 ° C (decomposition)

Analysis:

Calcd .: C 56.26 H 6.54 N 10.58 O 26.59 Found: C 56.31 H 6.50 N 10.37

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 20 a) 1,6-bis- {4- [2,5,8,11-tetrakis (tert-butoxycarbonylmethyl) -2,5,8,11-tetra azacyclododecyl] benzyloxy} hexane

3.33 g (2.16 mmol) of the compound according to Example 7a) are added in methanol 500 mg of 5% platinum / carbon hydrogenated to hydrogen saturation. It will 205 ml of hydrogen added. After filtering off the catalyst the substance before analysis. After the solvent has been stripped off, 3.19 g remain (95% of theory) of a colorless oil.

Analysis:

Calc: C 65.00 H 9.22 N 7.21 O 18.55 Found: C 64.79 H 9.23 N 7.16

b) 1,6-bis- {4- [2,5,8,11-tetrakis (carboxymethyl) -2,5,8,11tetraazacyclododecyl] - benyloxy} hexane

Analogously to the procedure in Example 7b, the title compound is obtained in 91% yield from the compound a) as colorless crystals.
Melting point: 162-165 ° C

Analysis:

Calc: C 56.61 H 7.12 N 10.15 O 26.10 Found: C 56.57 H 7.03 N 10.21

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 21 a) 1,6-bis - {- [2,5,8,11-tetrakis (tert-butoxycarbonylmethyl) -2,5,8,11-tetra azacyclododecyl] methoxy} hexane

4.1 g (2.95 mmol) of the compound according to Example 14a are added in methanol 500 mg of 5% platinum / carbon hydrogenated to hydrogen saturation. It will 268 ml of hydrogen added. After filtering off the catalyst the substance before analysis. After the solvent has been stripped off, 3.88 g remain (94% of theory) of a colorless oil.

Analysis:

Calc: C 61.77 H 9.64 N 8.00 O 20.57 Found: C 62.14 H 9.57 N 8.04

b) 1,6-bis - {- [2,5,8,11-tetrakis (carboxymethyl) -2,5,8,11-tetraazacyclododecyl] - methoxy} hexane

Analogously to the procedure, the title compound was obtained in 76% yield from the compound a) as colorless crystals.
Melting point: 110-112 ° C

Analysis:

Calc: C 50.51 H 7.41 N 11.78 O 30.28 Found: C 50.26 H 7.34 N 11.58

The gadolinium complex and its salts are obtained quantitatively for Example 1j described.

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 22 a) 1,2-bis- {4- [2,5,8,11-tetrakis (tert-butoxycarbonylmethyl) -2,5,8,11-tetra azacyclododecyl] benzyloxy} ethane

15.0 g (21.46 mmol) of the compound from Example 1j are dissolved in 200 ml of acetone with 2.31 g (10.7 mmol) of dibromoethane, 1.52 g (11.0 mmol) of potassium carbonate and 0.2 g of potassium iodide was refluxed for 10 hours, then concentrated and with Washed ethyl acetate / saline. After drying the organic phase and Chromatography on silica gel (ether / hexane) contains 12.36 g of pure analytical material Product as a colorless oil. (77% of theory)

Analysis:

Calc: C 64.23 H 9.02 N 7.49 O 19.25 Found: C 63.56 H 8.94 N 7.53

b) 1,2-bis- {4- [2,5,8,11-tetrakis (carboxymethyl) -2,5,8,11-tetraazacyclododecyl] benzyloxy} ethane

7.76 g (5.19 mmol) of the compound according to a) are, as in Example 1j wrote, implemented and processed. 4.50 g of a white product are obtained crystalline material (83% of theory).

Analysis:

Calc: C 55.05 H 6.73 N 10.70 O 27.5 Found: C 55.30 H 6.77 N 10.57

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

Gd complex Na salt of the Gd complex N-methylglucamine salt of the Gd complex Example 23 a) 1,12-bis - {[2,5,8,11-tetrakis (tert-butoxycarbonylmethyl) -2,5,8,11-tetra azacyclododecyl] methoxy} -4,9-diaza-5,8-dioxododecomethylene

121.35 g (17.25 mmol) of the compound according to Example 9b are dissolved in 200 ml of toluene dissolved and mixed with the equimolar amount of triethylamine. Drips at 0 ° C then a solution of 1.33 g (8.6 mmol) of succinic acid dichloride, whereby ensure good cooling and effective stirring. Half an hour after When the addition is complete, boil briefly, cool and suck off any unusual Triethylamine hydrochloride. You wash several times with water, dry, narrow and chromatographed with ether / hexane. A colorless oil is obtained in a total of 11.23 g (86% of theory).

Analysis:

Calcd .: C 60.29 H 9.32 N 9.25 O 21.13 Found: C 60.11 H 9.28 N 9.24

b) 1,12-bis -} [2,5,8,11-tetrakis (carboxymethyl) -2,5,8,11-tetraazacyclododecyl] - methoxy} -4,9-diaza-5,8-dioxododecamethylene

As described for Example 1j, the free product is obtained in 80% yield Complexing agent.

Analysis:

Calcd .: C 49.61 H 7.19 N 13.15 O 30.04 Found: C 49.38 H 7.26 N 13.35

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

Gadolinium complex Sodium salt of the Gd complex N-methylglucamine salt of the Gd complex

Claims (16)

1. Compounds of the general formula I where q for the digits 0, 1 or 2, A for the group in which
m the digits 1, 2, 3, 4 or 5,
l the digits 0, 1, 2, 3, 4 or 5,
R ^{1 'contains} an optionally imino, phenyleneoxy, phenylenimino, amide, ester group (s), oxygen, sulfur and / or nitrogen atom (s) optionally by hydroxyl, mercapto, imino and / or amino group (s) substituted straight-chain, branched, saturated or unsaturated C₁-C₂₀-alkyl group, which in the end either a ring of the general formula II wherein A¹ represents the group (CH₂) _m -CH- (CH₂) _l ,
has a functional group or a macromolecule bonded via this functional group,
mean B, D and E, which are the same or different, each for the group with R ^{2 ′} in the meaning of hydrogen or R ^{1 ′} ,
k has the meaning of the digits 0, 1, 2, 3, 4 or 5,
n in the meaning of the digits 0 or 1,
X for the radicals -COOY or -PO₃HY with Y in the meaning of a hydrogen atom and / or a metal ion equivalent of an element of atomic numbers 21-29, 31, 32, 38, 39, 42-44 or 57-83
stand,
with the proviso that B, C and D each contain at least 2 and a maximum of 5 carbon atoms, and their salts with inorganic and / or organic bases or amino acids. 2. Compounds according to claim 1, characterized in that Y is hydrogen represents atoms. 3. Compounds according to claim 1, characterized in that at least 2 of the Substituents Y metal ion equivalents of at least one element of the Atomic numbers are 21-29, 42, 44 or 57-83. 4. Compounds according to claim 1, characterized in that at least two of the substituents Y at least metal ion equivalents of a radionuclide an element of atomic numbers 27, 29, 31, 32, 38, 39, 43, 49, 62, 64, 70 or 77. 5. Compounds according to claim 1, characterized in that R ^{1 '} for an optionally imino, phenyleneoxy, phenylenimino, amide, ester group (s), oxygen, sulfur and / or nitrogen atom (s) containing given if substituted by hydroxy, mercapto, imino and / or amino group (s) straight-chain, branched, saturated or unsaturated C₁-C₂₀-alkylene group, which in the end as a functional group in which
R and R 'are the same or different and each represents a hydrogen atom, a saturated or unsaturated C₁-C₂₀ alkyl radical optionally substituted by a phenyl group or a phenyl group. 6. Compounds according to claim 1, characterized in that R ^{1 '} for one over a Group connected ring of the general formula II is. 7. Compounds according to claim 1, characterized in that R ^{1 '} for an optionally imino, phenyleneoxy, phenylene-imino, amide, ester group (s), oxygen, sulfur and / or nitrogen atom (s) containing optionally substituted by hydroxy, mercapto, imino and / or amino group (s) straight-chain, branched, saturated or unsaturated C₁-C₂₀ alkylene group, which in the end has an antibody bound via a functional group or an antibody fragment . 8. Compounds of the general formula I ' whereinA for the group B ', D' and E ', which are the same or different, each for the group stand,
n, m, k, l, q and X have the meaning given in claim 1,
R ¹ 'has the same meaning as R¹, with the exception that no macromolecule should be attached to the functional group at the end of the C₁-C₂₀ alkylene group,
R ² 'represents hydrogen or R ¹ ',
with the measure that B ', C' and D 'each contain at least 1 and a maximum of 5 carbon atoms and that at least two of the substituents Y are metal ion equivalents of at least one element of the atomic numbers mentioned in claim 1, and their salts with inorganic and / or organic bases or amino acids. 9. Pharmaceutical compositions containing at least one physiologically tolerated Liche compound according to claim 1 to 8, optionally with those in the Galenik usual additives. 10. Use of at least one physiologically compatible compound according to claim 3 for the preparation of agents for NMR or X-ray Diagnosis. 11. Use of at least one physiologically compatible compound according to claim 4 for the manufacture of agents for radio diagnostics. 12. Use of at least one physiologically compatible compound according to claim 4 for the preparation of agents for radio therapy. 13. Use of at least one physiologically compatible compound of general formula I 'according to claim 8 as hapten for the preparation of Antibodies. 14. Process for the preparation of compounds of general formula I. where q for the digits 0, 1 or 2, A for the group in which
m the digits 1, 2, 3, 4 or 5,
l the digits 0, 1, 2, 3, 4 or 5,
R ^{1 '} an optionally imino, phenyleneoxy, phenylenimino, amide, ester group (s), oxygen, sulfur and / or nitrogen atom (s) optionally containing by hydroxy, mercapto, imino and / or Amino group (s) substituted straight-chain, branched, saturated or unsaturated C₁-C₂₀ alkylene group, which in the end is a ring of the general formula II wherein A¹ represents the group (CH₂) _m -CH- (CH₂) _l ,
has a functional group or a macromolecule bonded via this functional group,
B, D and E which are the same or different, each for the group With
R ^{2 '} in the meaning of hydrogen or R ^1' ,
k has the meaning of the digits 0, 1, 2, 3, 4 or 5,
X for the radicals -COOY or -PO₃HY with Y in the meaning of a hydrogen atom and / or a metal ion equivalent of an element of atomic numbers 21-29, 31, 32, 38, 39, 42-44 or 57-83
stand,
with the proviso that B, C and D each contain at least one and a maximum of 5 carbon atoms and their salts with inorganic and / or organic bases or amino acids,
characterized in that in a manner known per se in compounds of the general formula III whereinA for the group B ', D' and E ', which are the same or different, each for the group where R ^{1 '} optionally containing an imino, phenyleneoxy, phenylenimino, amide, ester group (s), oxygen, sulfur and / or nitrogen atom (s), optionally by hydroxy, mercapto, imino and / or amino group (s) substituted straight-chain, branched, saturated or unsaturated C₁-C₂₀ alkylene group which at the end either a ring of the general formula II ' where R ^{2 ′} ′ is hydrogen or R ^{1 ′} ′
or mean a functional group,
X 'for the radicals -COOY' or PO₃Y 'with Y' are an acid protecting group
the protective group Y 'is split off and the acids of the formula III' thus obtained with Y 'in the meaning of a hydrogen atom, if desired
a) in a manner known per se with at least one metal oxide or metal salt of an element or atomic numbers 21-29, 31, 32, 38, 39, 42-44, 49 or 57-83 and then, if desired, existing acidic hydrogen atoms substituted by cations of inorganic and / or organic bases or amino acids, or
b) in a manner known per se with at least one metal oxide or metal salt of an element or atomic numbers 21-29, 31, 32, 38, 39, 42-44, 49, or 57-83 and then the metal complexes thus obtained in itself binds in known manner via the functional group contained in R ¹ 'to a macromolecule and, if desired, existing acidic hydrogen atoms are substituted by cations of inorganic and / or organic bases or amino acids, or
c) binds to a macromolecule in a manner known per se via the functional group contained in R ¹ ′ and then in a manner known per se with at least one metal oxide or metal salt of an element or atomic numbers 21-29, 31, 32, 38, 39, 42-44, or 57-83 and then, if desired, the acidic hydrogen atoms present are substituted by cations of inorganic and / or organic bases or amino acids. 15. The method according to claim 14, characterized in that the coupling of the macromolecule to the functionalized complex or ligand and, in In the case of coupling to the ligand, the subsequent complexation with the desired metal ion (s) is carried out on a stationary phase. 16. A process for the preparation of the pharmaceutical composition according to claim 9, characterized in that the in water or physiological saline dissolved or suspended complex compound, optionally with those in the Galenics usual additives, in one for enteral or parenteral appli cation brings appropriate form.