HU221187B1 - Process for the production of 1,4,7,10-tetraazacyclododecane derivatives, complexes and conjugates with antibody thereof and medicaments containing the same and diagnostics compraising these conjugates and complexes - Google Patents

Abstract

The present invention relates to compounds of formula (III) for the preparation of their pharmaceutically acceptable salts, wherein Q is CH2-CO2R, where R is hydrogen or alkyl of 1 to 4 carbon atoms, n is 0 to 4, R2 is nitro, amino or isothiocyanate. The invention further relates to a method for the preparation of the above compounds with metal ions complexes, compounds or complexes with conjugates with antibodies, and pharmaceutical compositions containing the complexes or conjugates. ŕ

Description

This invention relates to 1,4,7,10-tetraaza-cyclododecane derivatives, their complexes and antibody conjugates, to pharmaceutical compositions containing them, and to diagnostic compositions containing the complexes and conjugates.

Functional chelates or bifunctional coordinators are known to bind covalently to antibodies specific for cancer, cancer cell epitopes, or antigens. Radioactive nucleus complexes of such antibody / chelate conjugates are suitable for diagnostic and / or therapeutic use because they are capable of transporting the radioactive nucleus to a cancer cell [Meares et al., Anal. Biochem. 112: 68-78 (1984); and Krejcarek et al., Biochem and Biophys. Gap. Comm. 77, 581-585 (1977).

Aminocarboxylic acid chelating agents are known and have been the subject of research for many years. Examples are: nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), hydroxyethyl ethylenediamine triacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), trans-1,2-diamino -cyclohexane tetraacetic acid (CDTA) and 1,4,7,10-tetraaza-cyclododecane tetraacetic acid (DOTA). Various bifunctional chelating agents have been described and prepared which are based on aminocarboxylic acid. For example, the cyclic dianhydride of DTPA [Hnatowich et al. Science 220: 613-615 (1983), U.S. Patent 4,479,930] and mixed carboxylic anhydrides of DTPA (U.S. Patent Nos. 4,454,106 and 4,472,509) and Krejcarek et al., Biochem. and Biophys. Gap. Comm., 77, 581-585 (1977). When anhydrides are attached to proteins, coupling occurs via an amide bond, leaving four of the original -5-carboxymethyl groups on the diethylene triamine (DETA) backbone (Hnatowich et al., Int. J. Appl. Isot. 33: 327-332 (1982)]. U.S. Patent Nos. 4,432,907 and 4,352,751 also disclose bifunctional chelating agents useful for attaching metal ions to "organic groups such as organic target molecules or antibodies". As described above, the coupling here also takes place via the amide group of the diamine tetraacetic acid dianhydrides. Examples of such anhydrides are dianhydrides of EDTA, CDTA, propylene diamine tetraacetic acid and phenylene-1,2-diamine tetraacetic acid. U.S. Pat. No. 4,647,447 discloses various complex salts obtained from the anion of a complexing acid and used for various diagnostic purposes. It is taught that the coupling takes place via the carboxyl group of the complexing acid, which forms an amide bond.

Paik et al. [J. of Radioanalytical Chemistry 57, (12), 553-564 (1980)] discloses the use of p-nitrobenzyl bromide with a "blocked" diethylenetriamine (e.g., bis (2-phthalimidoylethyl) amine) followed by the deblocking process and the carboxymethylation using chloroacetic acid to give N'-p-nitrobenzyl diethylenetriamine-N, N, N, N-tetraacetic acid are described. In this method as well, since the bonding is through nitrogen, a tetraacetic acid derivative is obtained. Also disclosed are coupling of a bis-functional chelating agent and chelation with indium. Nitrogen substitution is also described (Eckelman et al., J. of Pharm. Sci. 64 (4), 704-706 (1975)], whereby amines such as ethylenediamine or diethylenetriamine are reacted with the corresponding alkyl bromide before carboxymethylation. The compound produced is an effective radioactive pharmaceutical imaging agent.

Another group of aminocarboxylic acid based bifunctional chelating agents are known. For example, Sundberg et al., J. Med. of Med. Chem. 17 (12), 1304 (1974)] discloses bifunctional analogs of EDTA. Representative examples of these compounds are 1- (p-aminophenyl) ethylenediaminetetraacetic acid and 1- (p-benzenediazonium) ethylenediaminetetraacetic acid. They also disclose the coupling of these compounds to proteins via the para-substituent and the attachment of radioactive metal ions to the chelating group. These compounds are also described in U.S. Patent Nos. 3,994,966 and 4,043,998, and in Biochemical and Biophysical Research Communications 75 (1), 149 (1977).

It is important to note that the attachment of the aromatic moiety to EDTA is via the carbon atom of the ethylene diamine backbone. Optically active bifunctional chelating agents of EDTA, HEDTA and DTPA are described in U.S. Patent No. 4,622,420. In these compounds, the aromatic moiety (which contains the functional moiety required for coupling to the protein) is linked by an alkylene moiety to the carbon atom of the polyamine, which contains the chelating moiety. Such compounds are also disclosed in U.S. Patent No. 4,647,447, International Patent Application WO 86/06384, and Brechbiel et al., Inorg. Chem. 25: 2772-2781 (1986).

Recently, certain macrocyclic bifunctional chelating agents and their copper chelate conjugates have been disclosed for diagnostic and therapeutic purposes in U.S. Patent No. 4,678,667 and to Moi et al., Inorg. Chem., 26, 3458-3463 (1987). The aminocarboxylic acid moiety is attached to the bifunctional chelating molecule via the ring carbon atom of the cyclic polyamine backbone. Thus, at one end, the linker moiety is linked to the ring carbon atom of the cyclic polyamine, while at the other end it is attached to a functional group capable of reacting with the protein.

Another notable group of bifunctional chelating agents are compounds in which the chelating moiety, i.e., the aminocarboxylic acid, is attached via a nitrogen atom to the functional group of the molecule, which molecule is also capable of reacting with the protein. so for example

HU 221 187 Β1

Mikola et al. (International Patent Application WO 84/03698) disclose a bifunctional chelating agent obtained by reacting p-nitrobenzyl bromide with DETA and treating the resulting compound with bromoacetic acid and thus converting it to aminocarboxylic acid. The nitro group is reduced to the corresponding amine group and then converted to the isothiocyanate group by reaction with thiophosgene. These compounds are bifunctional chelating agents capable of chelating with lanthanides, which can then be conjugated with diororganic molecules and used as diagnostic agents. After the linker moiety of the molecule is attached via one of the nitrogen atoms of the aminocarboxylic acid, a potential aminocarboxylic acid group is lost for chelation. In this way, DETA-based bifunctional chelates are obtained which contain four (not five) acid groups. In this way, this group of bifunctional chelating agents is similar to those in which binding to the proteins occurs via the amide group and so also a carboxyl chelating group is lost.

A recent publication by Caney, Rogers and Johnson [3rd. International Conference on Monoclonal Antibodies Fór Cancer; San Diego, Califomia 2 / 4-6 / 88, titled "Absence of Inrinsically Higher Tissue Uptake from Indium-111 Labeled Antibodies: Coadministration of Indium-111 and Iodine-125 Labeled B72.3 in a Nude Mouse Model" and "Influence of Chelator Denticity on the Biodistribution of Indium111 Labeled B72.3 Immunoconjugates in Nude Mice, "which describes the biodistribution of indium-111 complexed by EDTA and DTP A with bifunctional chelating agents. The aromatic ring is attached to the EDTA / DTPA moiety via an acetate methylene group. DK Johnson et al. Also recently described EDTA and DTPA bifunctional derivatives (Florida Conf. On Chem. In Biotechnology, April 26-29, 1988, Palm Coast, FL), where the p-isothiocyanato-benzyl moiety is one of the carboxymethyls. is attached via a methylene carbon atom. Hunt et al., U.S. Patent Nos. 4,088,747 and 4,091,088, previously disclosed ethylene diamine diacetic acid (EDDA) based chelating agents, wherein the aromatic ring to the EDDA moiety is an alkylene or an acetate methylene moiety. connected through a group. These compounds are suitable for study of hepatobiliary function as dormants. The preferred metal is technetium99m. Indium-111 and indium-113m are also preferred radioactive nuclei for imaging.

It is an object of the present invention to provide complexes which do not dissociate easily, which are capable of rapidly excreting the entire body except for the desired tissues, and to provide conjugates of these complexes with antibodies which produce the desired effect.

The figures of the present invention relate to: Figures 1-7. Figures ¹⁵³ Sm biodistribution are shown that had been added as a conjugate of the invention containing ¹⁵³ Sm. CC49-IgG antibody was used in the conjugate. Biodistribution was determined in nude mice bearing LS 174-T tumor.

8-14. Figures ¹⁵³ Sm biodistribution shown, which was added as a conjugate of the invention containing ¹⁵³ Sm. The conjugate contains the CC49-F (ab ') 2 antibody fragment. Biodistribution was determined in nude mice bearing LS 174-T tumor.

Surprisingly, it has been found that the complexes and / or conjugates of the invention are relatively stable (i.e., not easily dissociated) and some are excreted very rapidly from the body and from certain non-target organs such as the liver, kidney and bone.

It is within the scope of the present invention to provide novel bifunctional chelating agents, each containing a chelating function and a chemically reactive moiety which is capable of covalently linking it to a biomolecule. Also within the scope of the invention is the preparation of various bifunctional coordinator (BFC) metal complexes and coupling these complexes to antibodies to obtain radiolabeled antibodies and / or fragments such as samarium-153, lutetium-177 and yttrium-90. for diagnostic and / or therapeutic purposes.

The present invention relates to novel bifunctional chelating agents which form complexes with metal ions, particularly "radioactive" metal ions of the rare earth type. Rare earth metal metal ions useful in the present invention include La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y and Se, in particular Sm, Ho, Y, and Lu preferred radioactive rare earth-type metal ions include:., ¹⁵³ Sm, ¹⁶⁶ Ho, ^9U Y, ^L49 Pm, ¹⁵⁹ Gd, ^L40 La, ⁷⁷ Lu,> ⁷⁵ Yb, ⁴⁷ Sc, ¹⁴² Pr, especially ¹⁵³ Sm , ¹⁶⁶ Ho, ⁹⁰ Y and ¹⁷⁷ Lu. Other radioactive metal ions which may also be used are: ⁴⁷ Sc, ^99m Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ^'97 Pt, ^6,7 Cu, ^'98 Au, ¹⁹⁹ Au, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹ 'In, ^113m In, ^117m Sn, and ²¹² Pb / ²¹² Bi. The resulting complexes are covalently linked to an antibody or fragment thereof and the conjugates thus obtained can be used for therapeutic or diagnostic purposes. The complexes and / or conjugates can be used either in vivo or in vitro. The conjugates of the invention are preferably used for the treatment of cancers of mammals, especially human subjects.

Complexes and / or conjugates of the invention containing non-radioactive metal may also be used for diagnostic purposes and / or for the treatment of diseases such as cancer. Such use of non-radioactive metals causes radio frequency hyperthermia (Jpn. Kokai Tokyo Koho JP 61, 158,931) and fluorescence immuno-guided therapy (FIGVS) [K. Petterson et al., Clinical Chemistry 29, (1), 60-64 (1983) and C. Meares et al., Acc. Chem. 17, 202-209 (1984)].

More particularly, the present invention relates to the preparation of compounds of formula (III)

HU 221 187 Β1

Q is CH ₂ CO ₂ R, wherein

R is hydrogen or C 1-4 alkyl, n is a number from 0 to 4,

R ² is nitro, amino or isothiocyanate.

Also included within the scope of the invention are pharmaceutically acceptable salts of the compounds of formula (III) above.

Preference is given to compounds of formula III wherein R is hydrogen, n is an integer from 0 to 4, and R ² is nitro, amino or isothiocyanate.

If desired to prepare a conjugate of the invention can be different from R ² nitro groups to be.

The invention further relates to complexes with rare-earth metal-type metals, in particular complexes containing neutral radioactive or charged rare-earth metal-ion, and conjugates of these complexes with antibodies or antibody fragments. The present invention also encompasses compositions comprising these conjugates and pharmaceutically acceptable carriers therefor, particularly preferably liquid carriers. These conjugates and pharmaceutical compositions containing them may be used for diagnostic purposes or for the treatment of diseases, particularly cancerous diseases, comprising administering to mammals an effective amount of the compositions.

As used herein, the term "mammal" refers to any mammal that feeds on its little milk, preferably warm-blooded mammals, more preferably human. The term antibody includes any polyclonal, monoclonal, or chimeric antibody, or hetero-antibody, preferably a monoclonal antibody. The antibody fragment may be a Fab fragment and an F (ab ') 2 fragment, or any antibody portion having specificity for the desired epitope or epitopes. The metal chelate / antibody conjugate or antibody moiety includes the entire antibody and / or antibody fragment, including their metal synthetically or genetically engineered variants.

As used herein, the term "complex" refers to a compound comprising a compound of formula (III) of the invention containing a rare metal type ion, in particular a radioactive rare metal type metal complex, in which at least one metal atom is complexed. The term radioactive metal ion chelate / antibody conjugate or radioactive metal ion conjugate refers to a radioactive metal ion conjugate that is covalently attached to an antibody or antibody fragment. Radioactive expression when used together with the metal ion, it relates to the rare earth type elements isotopes which particles and / or photons are able to emit, for example, consisting of ¹⁵³ Sm, ^L66 Ho, ⁹ θΥ, ^L49 Pm, i5 ⁹ Gd, ^L40 La , ^'77 Lu, ¹⁷⁵ Yb, ⁴⁷ Sc, and ¹⁴² Pr. The terms bifunctional coordinator, bifunctional chelating agent, and functionalized chelate are equivalent to compounds containing a moiety capable of binding a metal ion in the form of a chelate, and it contains a linker group covalently linked to the chelate moiety which serves to covalently attach the molecule to the antibody or antibody fragment.

Pharmaceutically acceptable salts of the compounds of formula (III) are those which are non-toxic and useful in the therapy or diagnosis of mammals. Such salts are prepared in known manner, for example by reaction with organic or inorganic compounds, for example, sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, colic acid, palmic acid. , glutamic acid, d-camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, formic acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid, or other suitable acid. Pharmaceutically acceptable salts include salts with basic compounds, such as salts with any organic or inorganic basic compound, such as ammonia, alkali metals, alkaline earth metals or the like. Particularly preferred salts are the potassium, sodium, ammonium or mixtures thereof.

Of course, the compounds of formula (III) may be in the free acid form or in the protonated form, e.g. when the carboxylate group is protonated and / or when the nitrogen atom is protonated, e.g. when the HCl salts are prepared.

The bifunctional chelating compounds of formula (III) described above are capable of binding chelated metal ions, particularly radioactive rare earth metal ions, in the form of chelates to form metal ion chelates (also referred to as complex compounds). These complexes are capable of attaching to reactive carriers, such as reactive polymers, by virtue of the presence of a reactive group, or preferably when R ² is other than nitro in the complex of formula (III), these compounds are capable of covalently bonding to proteins or more preferably antibodies or antibody fragments. . Accordingly, the compounds of formula (III) are capable of complexing rare-earth metal ions, in particular radioactive rare-earth metal ions, and the resulting complexes are covalently bound to antibodies or antibody fragments, which are termed conjugates.

Antibodies or antibody fragments useful in the preparation of such conjugates are prepared in a known manner. For example, high specificity monoclonal antibodies can be prepared by hybridization techniques such as those described by Kohler and Milstein (1975, Naturre 256: 495-497; and Eur. J. Immunol. 6: 511-519 (1976)]. The antibodies thus obtained are generally highly specific

EN 221 187 Β1 capacity. The conjugates containing the radioactive metal ion can be used as antibodies to any desired antigen or hapten. Preferably, monoclonal antibodies or antibody fragments having high specificity for the desired epitope or epitopes are used. For example, antibodies to tumors, bacteria, fungi, viruses, parasites, mycoplasma, differential or other cell membrane antigens, pathogenic surface antigens, toxins, enzymes, allergens, drugs, and any biologically active molecule may be used in the present invention. Examples include the following antibodies or antibody fragments: CC-11, CC-46, CC-49, CC49 F (ab ') 2, CC-83, CC-83 F (ab') 2 and B72.3 [see D. Colcher et al., Cancer Slit. 48, 4597-4603 (August 15, 1988)] are antibodies CC-49, CC-83 and B72.3. The B72.3 hybridoma cell line is deposited with the American Type Culture Collection (ATCC) under the number HB 8108. The various CC-labeled antibodies are described in U.S. Patent Application No. 7-073685, filed July 15, 1987, and are available from NTIS. Other rat monoclonal antibodies bind to TAG72 tumor-associated antigenic epitopes. For a full list of antigens, see, for example, U.S. Patent 4,193,983. The radioactive metal ion conjugates of the present invention are particularly useful in the treatment and diagnosis of various cancers.

The preferred rare earth metal complexes (lanthanides or pseudo-lanthanides) of the present invention are represented by the following general formula:

C [Ln (BFC)] (IX) wherein Ln is a rare earth (lanthanide) ion such as Ce ³⁺ , Pr ³⁺ , Nd ³⁺ , Pm ³⁺ , Sm ³⁺ , Eu ³⁺ , Gd ³⁺ , Tb ³⁺ , Dy ³⁺ , Ho ³⁺ , Er ³⁺ , Tm ³⁺ , Yb ³⁺ and Lu ³⁺ , or pseudo-lanthanide metal ions such as Sc ³⁺ , Y ³⁺ and La ^{3 +} , particularly preferred metal ions are Y ³⁺ , Ho ³⁺ , Lu ³⁺ or Sm ³⁺ ; BFC is a bifunctional chelator; C is a pharmaceutically acceptable ion or ions that have sufficient charge to neutralize the complex.

If the BFC contains four or more of the group of negative charged moiety, then C is a cation or kationcsoport following: H ^+, Li ^+, Na +, K +, Rb ^+, Cs +, Mg ^2+, Ca ^2+, SR ² +, ba ^2+, NH 4 ^+, N (CH 3) ₄ +, N (C ₂ H _{5) 4} ^+, N (C3H7) 4+, N (C4H9) 4 ^+, As (C 6 H _{5) 4} ^+, [(C ₆ H ₅ ) 3 P =] 2N + or other protonated amine groups. If the BFC group contains three negatively charged portions, the presence of C is not required. When the BFC group contains two negatively charged moieties, C represents an anion F ~, CL, Br ~, I ~, C1O ₄ ~, BF ₄ , h ₂ po ₄ -, hco ₃ -, hco ₂ -, ch ₃ so ₃ -, h, cc ₆ ii ₄ SO ₃ , PF ₆ -, CH ₃ CO ₂ and B (C ₆ H ₅ ) ₄ .

The conjugates and, in some cases, the complexes of the invention are also used in the form of compositions. These compositions contain a compound of Formula III, an antibody and / or a metal ion, and a physiologically acceptable carrier or excipient. For example, a composition of the invention may comprise a physiologically acceptable carrier, a complex (metal ion + ligand), a conjugate (metal ion + ligand + antibody) or (ligand + antibody). The preparation of such compositions may be carried out in a manner known per se, and may take the form of suspensions, injectable solutions or other suitable forms. The physiologically acceptable suspending medium may be used with or without various additives.

The compositions of the present invention are in solid or liquid form which contain an active radioactive core complexed with a ligand. These compositions may be in kit form in which the two components (i.e., ligand and metal, complex and antibody, or ligand / antibody and metal) are mixed together at the appropriate time before use. The composition also comprises a carrier, either pre-mixed or in kit form, which is generally pharmaceutically acceptable.

Injectable compositions obtained by the process of the invention may be in the form of suspensions or solutions. In the preparation of various formulations, it is generally found that the water solubility of the salt form compounds is higher than that of the acid form compounds. For solution formulations, the complexes (or separate components) are dissolved in a physiologically acceptable carrier. Such carriers include, for example, a suitable solvent, a preservative such as benzyl alcohol, or a buffer, if necessary. Usually the solvent used is water, aqueous alcohols, glycols, phosphonate or carbonate esters. The aqueous solutions contain no more than 50% by volume of organic solvent.

Injectable suspensions of the present invention generally contain a liquid suspending medium to which various additives may be added. The suspending agent may be, for example, aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or high purity mineral oils, or aqueous carboxymethylcellulose. For example, suitable physiologically acceptable additives are needed to keep the complexes in suspension. For example, thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin or alginates may be used. Various surfactants may also be used as suspending agents, such as lecithin, alkylphenol, polyethylene oxide adducts, naphthalene sulfonates, alkylbenzene sulfonates, or polyethyleneoxy sorbitan esters.

Various materials are used to influence hydrophilization, density, surface tension, which in some cases facilitate the preparation of injectable suspensions. For this purpose, for example, silicone antifoam, sorbitol or sugars are used.

For therapeutic purposes, an "effective amount" of the compositions is used. The dose required will generally depend on

EN 221 187 Β1 of the disease to be treated. The compositions are generally used for in vitro diagnosis, but may also be used for in vivo diagnosis. The conjugates or compositions of the present invention may also be used for radioimmuno-controlled surgical purposes (RIGS), for example, the following metals: 99mTc, Lilin, 113mln, 67Ga or 68Ga.

The chelates of the invention may also be used for various other purposes, such as magnetic resonance imaging, such as Gd ⁺³ complexes of compounds of formula III, as well as in various polymeric carrier forms, for example as a diagnostic agent, and lanthanide metal or pseudo- lanthanide metal ion removal by selective extraction.

Many of the chelating agents, complexes and antibody conjugates of the present invention are much more stable and / or have a better biodistribution and / or faster elimination from the body than other similar formulations known hitherto. The invention also relates to processes for the preparation of these chelating agents, complexes and antibody conjugates.

The 12-membered macrocyclic bifunctional chelating agents of formula (III) are preferably and generally prepared by monofunctionalizing the free base macrocyclic compound (e.g., 1,4,7,10-tetraazacyclododecane) with the corresponding electrophilic compound (e.g. , an appropriately substituted α-halo carboxylic acid ester). This electrophilic compound must contain a suitable linker moiety or a suitably protected linker moiety that permits covalent attachment of the bifunctional ligand to the protein, antibody, or antibody fragment.

It is known in the art that alkylation of mono-N-functionalized polyazacrocyclic compounds results in a mixture of different, hardly separable products [T. Kaden, Top Curr. Chem. 121: 157-75 (1984)]. This was eliminated by the described methods by using a large excess of the macrocyclic compound (5-10 equivalents relative to the electrophilic compound), which favored the formation of the monoalkylated adduct [M. Studer and T. A. Kaden, Helv. Chim. Acta 69: 2081-86 (1986); E. Kimura et al., J. Chem. Soc. Chem. Commun. 1158-59 (1986)].

Other methods include the preparation of mono-N-functionalized polyazacrocyclic compounds using lengthy protective, functionalizing and deprotection reactions (P, S, Pallavicini et al., 1987, J. Amer. Chem. Soc. 109, 5139-44; M. F. Tweedle et al., Eur. Pub. Pat. Appl. No. 0232-751]. Recently, reductive amination of substituted phenylpyruvic acids and amines has been reported in an extract from a recent meeting of the Abbott Laboratory [D. K. Johnson et al., Florida Conf. On Chem. In Biotechnology, April 26-29, 1988, Palm Coast, FI]. It is also known to prepare a mono-N-alkylated polyazacrocyclic compound by applying 1 to 5 equivalents of the electrophilic compound to the corresponding macrocyclic compound in a solvent that does not promote proton transfer (U.S. Pat. No. 289,163). United States Patent Application, filed December 22, 1988).

I-II. The following Schemes illustrate the general preparation of specific compounds for the chelating agents of the present invention using the appropriate electrophilic and polyazacrocyclic compounds at different stoichiometric ratios, temperatures and concentrations in a suitable organic solvent. As the organic solvent, any hydrocarbon compound can be used which provides the appropriate solubility, such as acetonitrile, isopropanol, methylene chloride, toluene, chloroform, n-butanol, carbon tetrachloride, tetrahydrofuran, 5% ethanol chloroform, particularly preferred are chloroform, methylene chloride, n-butanol, 1,4-dioxane and acetonitrile. The macrocyclic and electrophilic compounds are used in stoichiometric or near stoichiometric ratios to give the corresponding mono-N-alkylated product in a single step. The temperature is generally from 0 ° C to the reflux temperature of the solvent, preferably from 0 ° C to 25 ° C. The reaction time is usually 1 to 24 hours.

II. The α-haloic acid esters are required to carry out the reaction scheme. A suitable process is the bromination or chlorination of acid halides formed in situ [D. N. Harpp et al., J. Org. Chem., 40, 3420-27 (1975)]. In this method, it is possible to produce only α-halogenated alkanoic acids which even contain a reactive benzyl group. In the general preparation of substituted acid halides, the organic acid is reacted with thionyl chloride or sulfuryl chloride [E. Schwenk et al., J. Amer. Chem. Soc., 70, 3626-27 (1944). In both methods, free carboxylic acid is used as a starting material, which is commercially available.

Polyazamacrocyclic compounds such as

1,4,7,10-tetraaza-cyclododecane can be prepared according to known methods (e.g. T. J. Atkins et al., J. Amer. Chem. Soc. 96, 2268-70 (1974) and T. J. Richman et al., Org. Synthesis 58, 86-98 (1978)].

The carboxymethylation of the mono-N-functionalized macrocyclic compound can be accomplished by the Desreux method using a bromoacetic acid derivative and a suitable base [J. F. Desreux, Inorg. Chem., 19, 1319-24 (1980)].

Any starting material used in the process of the invention is commercially available or can be prepared according to known methods described in the literature.

Although the reaction schemes relate to the preparation of one particular compound, other compounds of the formula n = 0-5 may of course be prepared.

The use of optically active alkylating agents minimizes the formation of diastereomers and simplifies the

EN 221 187 Bleeding the procedure. The separation of the individual diastereoisomers increases the amount of each of the easily purified lanthanide complexes desired for radiolabeling and antibody conjugation.

The electrophilic compound can also be prepared in a known manner. Such methods are described, for example, in Acc. Chem. 17, 202-209 (1984). Compounds of formula (III) wherein R ² is amino are obtained by reduction of the corresponding compounds of formula (III) wherein R ² is nitro. The preparation of compounds of formula III wherein R ² is isothiocyanate is carried out by reacting an amino chelate [R ² is amino] with thiophosgene (U.S. Patent No. 289,172, filed December 23, 1988). on)

The radio package can be obtained in various ways, for example by bombarding the nucleus with neutrons in a nuclear reactor, for example:

Sm-152 + neutron + Sm-153 gamma

Alternatively, the radio packages are obtained by bombarding the nucleus with a linear accelerator or cyclotron. In another method, the radio packages are separated from a mixture of fusion products. The method of preparing the radioactive nuclei is not critical to the compounds of the invention.

The conjugates of the invention may be prepared by first forming the complex and then binding to the antibody or antibody fragment. Thus, in this process, the ligand is first obtained, then complexed with the metal, and then the resulting complex is added to the antibody. Alternatively, in the preparation of labeled antibody conjugates, BFC is first bound to the antibody and then chelated to yield the radioactive BFC labeled antibody conjugate.

In the following examples, which illustrate the invention in greater detail, the following terms, experimental conditions, are used.

Mass spectral measurements were performed on a Finnigan TSQ mass spectrometer (mode Q1MS) or a VG ZAB-MS high resolution mass spectrometer (xenon fast atomization, 3: 1 = dithiothreitol: dithiol erythritol).

1 H and ¹³ C NMR spectra were recorded on a Varian VXR300, Bruker APC 300, IBM / Bruker NR-80 or Jeol FX400 spectrometer. All spectra were recorded at 30 ° C unless otherwise noted. The 1 H-NMR spectrum was recorded at 300 MHz, 80 MHz, or 400 MHz, depending on the device listed above: the ¹³ C-NMR spectrum was recorded at 75 MHz, 20 MHz or 100 MHz, also depending on the device. NMR values reported are U per TMS (tetramethylsilane) values or, if D2O is used as solvent, per DSS (2,2-dimethyl-2-silapentane-5-sulfonic acid, sodium salt).

Infrared (IR) spectra were recorded on a Nicolet 5SL FT / IR instrument.

Most of the solvents used in the chromatographic methods were Fisher HPLC grade. Ammonium acetate was purchased from Aldrich. The water was purified using the Barnstead NANPOpure ™ water purification system. Preparative chromatography of the organic compounds was carried out either by normal gravity chromatography or by flash chromatography [C. W. Still et al., J. Org. Chem., 43, 2923-24 (1978)]. The following solvent systems were used for the chromatography:

solvent components 1 CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 2: 2: 1 V: V: V 2 CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 12: 4: 1 V: V: V 3 CHCl ₃ : CH ₃ OH: Conc. NH ₄ OH 16: 4: 1 V: V: V 4 CHCl3: CH3OH: Conc. NH ₄ OH 4: 2: 1 V: V: V 5 CHCl3: CH3OH: Conc. NH ₄ OH 3: 2: 1 V: V: V 6 CHCl?: CH ₃ OH: conc. NH ₄ OH 7: 3: 1 V: V: V 7 brine (0.85% NaCl in distilled water): conc. NH ₄ OH 4: 1 V: V 8 CHCl3: CH3OH: Conc. NH ₄ OH V: V: V

The Rf values in the Examples were obtained using the above solvent systems and commercially available normal phase silica TLC plates (GHLF 250 microns, Analtech Inc. or Merck Kiesel gel 60F254). Merck 60, 60 A silica gel was used for preparative column chromatography.

The percentages given in the examples are by weight unless otherwise indicated.

In some cases, the resulting solid was dried on a rotary evaporator (Buchi 461) and / or in a vacuum oven at 55-60 ° C. In some cases, a Virti 10-010 automatic freeze dryer or a Speed VacTM concentrator was used to remove the solvent.

Samarium-1153 and Lutetium-177 were obtained from Research Reactor, Univer7

HU 221,187 Β1 sity of Missouri (Columbia, MO), Yttrium-90 metal was purchased from Oak Ridge National Laboratory.

1- (4-Isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid (SCN-Bz-DTPA) is described by M. W. Brechbiel et al., Inorg. Chem. 25, 2772-2781 (1986)] and the product was purified by anion exchange HPLC (Q-Sepharose ™) to give a single product. The reagents used are listed below and the places of purchase are in parentheses: N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES), free acid or sodium salt (Bering Diagnostics, La Jolla, CA), citrate (certified, Fisher Scientefic), thiophosgene (Aldrich Chemicals), ammonium acetate, sodium acetate, ethylenediaminetetraacetic acid, phosphate buffer (Sigma Diagnostics).

The HPLC column used was Hand-packed Q-Sepharose ™ (Pharmacia) or 1.5 cm x 25 cm or 2.5 cm x 25 cm; Zorbax ™ BIO Series GF-250 (9.4mm x 25cm) from Du Pont Instruments; Vydac ™ (trade name of Separation Group, Hesperia, CA) protein C-4 (4.6 mm x 25 cm) manufactured by Separation Group (Hesperia, CA); and Mono-QTM by SP-Sephadex ™ (trademark of Pharmacia Biotechnology Products), manufactured by Pharmacia. Sep-PakTM (Waters Associates trade name) C-18 pack, available from Waters Associates (Milford, MA), Sephadex ™ G25 Disposable Column (2.2 mL) from Isolab Inc. (Akron, OH) and CentriconTM-30 ( Amicon Division, a trademark of WR Grace & Co., Danvers, MA), available from Amicon.

Five different HPLC systems were used for analysis and sample separation:

System I: LKB 2150 Pump, 2152 Controller, UV Detector - LKB 2238 UV Cord, Barthold LB 506 A HPLC-Radioactivity Monitor (of the International Berthold Group) and Gilson Fraction Collector 201202 (Gilson International, Middleton, WI) ;

II. system: automatic sample dispenser, WISP 710 B, two pumps (model 510) and automatic gradient regulator (Waters Associates), Perkin-Elmer LC-75 spectrophotometric detector, Beckman model 170 radioisotope detector, and fraction collector (Frac-100, Pharmacia);

III. system: two Waters M-6000A pumps, Waters 660 solvent programmer, Waters Model 481 Variable Wawelength Detector, Waters Data Module, and ISCO fraction collector;

ARC. system: Dionex BioLCTM system with variable wavelength UV detector; and

System V: Waters Model 590 Pump with ISCO Model 2360 Gradient Programmer and Dionex Variable Wavelength Detector.

A Sorvall RT 6000B (refrigerated centrifuge, manufactured by Du Pont) was used for centrifugation and concentration. A VAC concentrator (Savant Instruments Inc., Hicksville, N.Y.) was used to remove volatile solvents. Radiological measurements were performed with a Capintec ™ Radioisotope Calibrator with CRC-12 or liquid scintillation (Beckman LS 9800, Beckman Instruments, Inc.,

Irvine, CA), or Canberra 35+ Multi-Channel Analyzer, 3 in. hollow crystalline sodium iodide with an internal partition.

In the examples for the preparation of complexes, the percentage of the complex was determined by the following known ion exchange method.

A 10 mL plastic column was filled with 1-2 mL of SP-Sephadex ™ C-25, previously swollen in water, and excess water was removed by pressure applied to the top of the column. 15 µl of the test solution was added to the resin at the top of the column and then 2 ml of a 4: 1 (v / v) isotonic saline: concentrated ammonium hydroxide eluant was added. The eluent was collected in a graduated tube. A further 2 ml of eluent was added. After collecting the eluent in a second tube, the resin was dried by applying pressure to the top of the column. The dried resin was then transferred to a third measuring tube. The activity of the measuring tubes was determined with a NaI counter connected to a Canberra multi-channel analyzer. The amount of metal bound in the complex was expressed as a percentage of the total activity determined in the eluent. The amount of uncomplexed free metal is expressed as a percentage of the total amount of metal remaining on the resin.

Removal of uncomplexed metal by ion exchange

A plastic column of 10 mL volume was filled with 0.5 mL of SP-Sephadex ™ C25 resin in water. Excess water was removed by centrifugation at 3000 rpm for 4 minutes. A solution of 200-500 Si complex compound was then added to the top of the resin and centrifuged again at 3000 rpm for 4 minutes. The uncomplexed metal remaining on the column and the complexed metal were removed by elution with solvent.

Preparation of yttrium complex

A complex compound was prepared by making 3 x 10 ⁴ molar aqueous solution of yttrium (YCl ₃ 6H ₂ O, 303.26 g / mol; or Y (OAc) 3, 12.1% H ₂ O). To the resulting solution was added radioactive Y3 + solution (Oakridge National Laboratories) to provide the desired radioactivity. Thereafter, 10 SiO ligand solution (0.03 mol) was added to 990 SiO3 + solution to provide a 1: 1 molar ligand: metal concentration. Ten times the ligand solution was used to provide a 10: 1 ligand: metal ratio. The pH was adjusted to 7.4 using microliters of hydrochloric acid or sodium hydroxide. The amount of complexed yttrium in the resulting solution was then determined using the cation exchange method described above.

Production of Samarium Complex

The samarium complex was prepared by the method described above for the preparation of the yttrium complex, except that a 3 χ 10 ⁴ molar solution of Samarium was prepared by dissolving 348.7 g / mol Sm ₂ O _{3 in} 0.1 M hydrochloric acid. The radioactive Sm-153 is a 3x10 ⁴ mol / 0.1 M hydrochloric acid solution

HU 221,187 was obtained from the University of Missouri Research Reactor, Columbia, Missouri.

Here's what the abbreviations used in the examples mean:

-OAc is an acetate group, -OCOCH ₃ ;

TLC refers to thin layer chromatography;

Dl H ₂ NANOpureTM is deionized water;

NANOpure ™ water is water purified by filtration produced by Barnstead NANOpure ™;

The ambient temperature is usually 20-25 ° C.

Overnight means 9-18 hours.

LC stands for liquid chromatography.

NBS means N-bromosuccinimide.

MES is 2- (N-morpholino) ethanesulfonic acid;

HPLC means high performance liquid chromatography;

PBS is phosphate buffered saline, manufactured by Sigma, 120 mM NaCl, 2.7 mM KCl and 10 mM phosphate buffer, pH 7.4;

SP-Sephadex ™ C-25 Resin means: a cation exchange resin with sulfonic acid functionalities, available from Pharmacia Inc.;

pD is the pH at which hydrogen is deuterated;

DOTA is 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid;

HEPES represents N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid;

BFC is a bifunctional chelator; cyclen is 1,4,7,10-tetraaza-cyclododecane; EA-DO3A is 1- [2- (4-aminophenyl) ethyl] 1,4.7.10-tetraaza-cyclododecane-4,7,10-triacetic acid;

DTPA is diethylene triamine pentaacetic acid;

SCN-Bz-DTPA is 1- (4-isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid;

EDTA is ethylenediaminetetraacetic acid; the chelator is the same as the ligand; the complex is the same as the chelate; the conjugate is a chelate or complex which is covalently linked to an antibody or antibody fragment; and the antibody may be CC-49, CC-83 and B72.3, and fragments thereof, such as Fab and F (ab ') 2. Other antibodies which may also be used have been mentioned above. The B-72.3 hybridoma cell line is deposited with the American Type Culture Collection under ATCC HB 8108, the other rat monoclonal antibodies referred to bind to TAG-72 tumor-associated antigenic epitopes.

The following examples are by no means limiting but merely illustrative of the invention.

Preparation of starting compounds

Example G 1- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane

Dissolve 3,5 g (20.3 mmol) of 1,4,7,10-tetraaza-cyclododecane in 50 ml of pentene-stabilized chloroform and add 4 g (17.4 mmol) of 1 - ( 2-bromoethyl) -4-nitrobenzene.

Stirring was continued for 18 hours at room temperature, whereupon the amine hydrobromide crystallized out of solution. The entire reaction mixture was applied to a silica gel flash chromatography column eluted with 5% methanol in chloroform. 200 ml of this solution were used as eluent and elution was continued with solvent system 3. 1.45 g (9.7 mmol) of p-nitrostyrene are isolated from the desired product (Rf = 0.98, solvent system 1). The desired monofunctionalized title compound was obtained as an orange oil (2.27 g, 7.06 mmol, 40.6%) which solidified (Rf = 0.73, solvent system 1).

A small sample of the title compound was recrystallized from CHCl ₃ / cyclohexane, m.p. 146.5-148.5 ° C. 1 H-NMR (CDCl ₃ )

8.135 (d, m), 7.395 (d, m), 2.91 (t), 2.77 (t), 2.72 (t),

2.50 (t), 2.60 (s); C-NMR (CDCl ₃ 75 MHz):

148.5, 146.7, 129.6, 123.4, 55.5, 51.4, 46.9, 45.9,

45,1,33,7.

Example II 1- (4-Nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane ml of chloroform was stirred in 3.5 g (20.3 mmol).

1,4,7,10-tetraaza-cyclododecane and 1.7 g (7.87 mmol) of p-nitrobenzyl bromide and the resulting mixture was stirred under nitrogen for 24 hours at 25 ° C. The chloroform suspension containing the hydrobromic acid salt was then subjected to flash chromatography (silica gel, solvent system 3) to give the title product (2.13 g, 6.93 mmol) as a pale yellow solid, analytical purity (88%, Rf = 0.58, solvent system 3), mp 128-129 ° C. 1 H-NMR (CDCl ₃ )

8.19 (d), 7.49 (d), 3.69 (s), 2.82 (t), 2.70 (t), 2.59 (m); C-NMR (CDCl ₃ , 75 MHz): 147.2, 128.4,

123,8,58,8,51,7,47,1,46,3,45,1.

Example J 1- (4-Aminobenzyl) -1,4,7,10-tetraaza-cyclododecane in methanol (690 mg, 2.25 mmol) was dissolved in 1- (4-nitrobenzyl) -1,4,7. 10-Tetraaza-cyclododecane (prepared as in Example H) was added to the solution and 350 mg of 10% palladium on carbon was added. Excess hydrogen was then passed through the solution at 25 ° C. After 45 minutes, TLC showed the title product to be formed (Rf = 0.33, solvent system 3). The title product is then purified by chromatography (silica gel, solvent system 3) to give 480 mg (77%) of the title compound as a pale yellow, clear oil.

The resulting free base (103 mg, 0.37 mmol) was converted to the hydrochloride salt by passing hydrochloric acid gas through the ethanolic solution. The resulting tetrahydrochloride salt was washed with cold ethanol and dried in vacuo to give 147 mg (0.347 mmol) of product.

M.p. 255-260 ° C (dec.). 1H (D2O, pD = 1.9)

7.56 (d), 7.47 (d), 3.95 (s), 3.28 (t), 3.23 (t), 3.09 (t),

2.96 (t); C-NMR (D2O, pD = 1.9, 75 MHz)

138.3, 134.2, 132.2, 126.0, 58.5, 50.3, 46.7, 44.6,

44.3.

Preparation of the final product ligands

Example 1 1- (4-Aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid 4,7,10-trimethyl ester 4 ml of argon purged acetonitrile are mixed.

100 mg (0.236 mmol) of 1- (4-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane tetrahydrochloride salt (prepared according to Example J), 260 mg (1.88 mmol) of potassium carbonate and mg (0.708 mmol) of methyl bromoacetate. The resulting mixture was stirred for 48 hours at 25 ° C and the saline solution applied to a 1 x 4 cm flash silica gel column and eluted with acetonitrile. The fractions containing the desired product were combined and the solvent was removed by rotary evaporation to afford 65 mg (0.132 mmol, 56%) of the title compound.

1 H-NMR (CDCl 3):

7.29 (d), 6.75 (d), 4.62 (s), 4.20 (s), 3.70 (s), 3.69 (s), 3.64 (s), 3.62 (t), 3.27 (s), 3.06 (t), 2.82 (broad t), 2.79 (broad t);

¹³ C NMR (CDCl 3, 75 MHz):

171.4, 171.0, 148.4, 132.8, 117.5, 115.0, 555.9,

55.3, 54.7, 53.1, 51.7, 51.4, 50.6, 50.5, 47.4.

Example 2 1- (4-Aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid hydrochloride salt in 2 ml of 6N hydrochloric acid at 80 ° C for 2 hours 1- (4-aminobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid 4,7,10-trimethyl ester (prepared as in Example 1) was heated. The TLC performed showed different products (Rf = 0.6, solvent system 1). After removal of the solvent, 41 mg of crude title product are obtained.

Example 3 1- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid 4,7,10-trimethyl ester 2.24 1- (2- (4-Nitrophenyl) ethyl) -1,4,7,10-tetraaza-cyclododecane (6.97 mmol) (prepared as in Example G) was dissolved in 75 mL of argon-flushed acetonitrile and added with vigorous stirring. 17 g of anhydrous potassium carbonate. To the resulting reaction mixture, 3.2 g (20.9 mmol) of methyl bromoacetate was added dropwise over 5 minutes and the mixture was stirred for 17 hours under argon. TLC analysis showed the conversion of starting material (Rf = 0.73, solvent system 1) to the new product (Rf = 0.46, solvent system 1). Chloroform (70 mL) was then added to the solution and the mixture containing the suspended salt added to a 1 x 7 inch flash silica gel column. The product was eluted with 10% chloroform in methanol to give the title compound (2.95 g) as an amber oil which, when dried in vacuo, gave a crumbly glassy material (78%).

1 H-NMR (CDCl 3):

8.17 (m, m), 7.4-7.66 (m), 2.38-3.95 (m).

13 C NMR (CDCl ₃ , 75 MHz):

171.5, 171.2, 146.7, 144.5, 130.3, 123.9, 56.0, 55.2,

53.5, 53.4, 52.6, 51.9, 51.8, 50.6, 48.1, 29.5.

Example 4 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid 4,7,10-trimethyl ester 50 ml 2.8 g of the compound of Example 3 are dissolved in methanol and 600 mg of 10% palladium on carbon are added to the resulting solution while stirring under nitrogen. The solution was kept under nitrogen and is bubbled hydrogen at 20-25 ° C for 10 ⁵ psi for 2.5 hours on. The solution was then purged with nitrogen for several minutes and the catalyst was removed by filtration through Celite. TLC analysis (10% methanol in chloroform) gave the title product (Rf = 0.14). The title compound was purified from silica gel eluting with chloroform to give 2.2 g (4.3 mmol, 83%) of product as a white glass.

1 H-NMR (CDCl ₃ ):

7.03 (d, m), 6.63 (d), 3.4-3.6 (m), 2.7-3.2 (m); ¹³ C-NMR (CDCl ³ , 75 MHz):

171.4, 171.2, 145.6, 129.7, 125.6, 115.5, 55.9, 54.4,

54.3, 53.0, 52.8, 51.8, 51.6, 50.3, 47.8, 28.6;

FAB MS m / e 508 [M + H + J +].

Example 5 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid hydrochloride salt; (EA-DO3A) ml of concentrated hydrochloric acid (850 mg, 1.69 mmol) was dissolved in ml of concentrated hydrochloric acid and the resulting solution was stirred at 70-80 ° C for 2.5 hours and then allowed to stir at 25 ° C. at this temperature, stirring was continued for 18 hours. The solvent was then removed in a rotary evaporator under reduced pressure (10 mm Hg, 75 ° C), and the solvate and excess hydrochloric acid were removed by drying under vacuum (10-1 mm, 45 ° C) from the resulting clear oily material. The title compound was obtained as a white solid, 890 mg, Rf = 0.46, solvent system 2.

1 H-NMR (D 2 O, pD = 1.9, T = 90 ° C)

7.50 (d), 7.41 (d), 4.26 (s), 3.43-3.68 (m), 3.0-3.3 (m);

¹³ C-NMR (D 2 O, pD = 1.9, T = 90 ° C)

176.7, 170.9, 139.8, 133.0, 131.2, 125.9, 57.5, 57.1,

55.4, 54.4, 52.7, 51.0, 50.6, 31.3;

FAB MS: m / e 466 [M + H +], 488 [M + Na +], 510 [M + 2Na ⁺ H +] +.

The HPLC analysis showed a purity of 92% (254 nm) with a particle size of 10 cm for the assay.

HU 221 187 Bl

OD53 RAC II reverse phase column, eluting with 10% acetonitrile in 0.05 M potassium phosphate buffer, pH 7 was used.

Example 6 1- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid;

(SCN-EA-DO3A)

Dissolve 106 mg (0.21 mmol) of the compound prepared in Example 5 in 2 mL of water under nitrogen with stirring and add sodium bicarbonate (105.6 mg, 1.26 mmol) to prevent the formation of carbon dioxide (pH = 8). , 0). Thiophosgene (16.8 µL, 0.22 mmol) was then added and, after vigorous stirring for 1 h, TLC (20% aqueous acetonitrile) was performed to give the starting material (Rf = 0.12) as the title compound. (Rf = 0.26). The solvent was then removed in a rotary evaporator to give 131 mg of the title compound containing sodium chloride. According to infrared analysis (KBr tablets) an isothiocyanate group is present, SCN = 215m cm ^-1 .

Example 7

- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododec-4,7,10-tri-acetic acid triethyl ester 5 ml of acetonitrile was mixed with 395 mg (1.12 mmol) of 1 [ 2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane (prepared according to Example G) and 1.5 g (11 mmol) of potassium carbonate, followed by addition of 496 μΐ (4.5 mmol) with stirring. ) ethyl bromoacetate. The mixture was then heated at 68 ° C for 1 hour under nitrogen and the resulting slurry was filtered, washed with CH ₂ CN (2 x 10 ^{8 x 10} mL), and the filtrate was concentrated to give the title compound as a viscous oil. ml of diethyl ether and concentrated. This gives 650 mg (100%) of the title compound.

1 H-NMR (CDCl ₃ ):

8.2 (d), 7.6 (d), 4.3 (q), 2.6-3.8 (m), 1.55 (t);

FABMS: [M + H] + = 588.

Example 8 1- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid

Example 7 (200 mg, 0.35 mmol) was suspended in water (15 mL), followed by the addition of 59 μΐ sodium hydroxide (50% w / w, 3 equivalents) and the resulting mixture was stirred at 80 ° C for 6 h. The resulting homogeneous orange solution was then cooled to room temperature and freeze-dried to give a tan solid. This material was purified by HPLC chromatography (Q-Sepharose ™, HPLC system III) eluting with a 0-10% aqueous HOAC linear gradient over 60 minutes to give 1- [2- (4) -nitrophenyl) ethyl] 1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid is obtained.

1 H-NMR (D 2 O)

8.22 (d), 7.55 (d), 3.60-3.90 (m), 3.10-3.40 (m).

Example 9 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododec-4,7,10-triacetic acid; (EA-DO3A)

Parr hydrogenated in 50 ml of water

214 mg (0.43 mmol) of the compound of Example 8 and 40 mg of 10% palladium on carbon are added and the resulting suspension is shaken until hydrogen uptake ceases. The product was filtered and the aqueous solution was freeze-dried to give the title compound (197 mg, 98%) as a beige solid.

1 H-NMR (D 2 O):

7.35 (d), 7.20 (d), 3.10-3.70 (m);

C-NMR (D2O)

173.85, 172.50, 137.90, 131.90, 130.22, 121.55, 56.25, 55.14, 54.42, 50,12,49,65,49,31, 31, 00th

Production of end products, complexes

The metal ligand complexes were prepared by various methods. In these methods, the metal and ligand were mixed in an aqueous solution while adjusting the pH to the desired value. The complexation was carried out in a solution containing salts and / or buffers and water. Occasionally, the solutions were heated to obtain a higher complex yield than when the complexation was carried out at ambient temperature. It was also found that the processing step in the synthesis of BFC compounds also influences complexation.

Stability of chelates as a function of pH

The stability of the metal ligand complexes was tested at various pH values by determining the amount of complexes in the solution. At low pH, metal release can occur due to protonation of the ligand. At high pH, metal hydroxides are also involved in chelation. Therefore, when researching inert chelates, it is necessary that the inert chelate remain stable at both high and low pH.

The stability of the chelates of the invention as a function of pH was tested according to the methods described in the following examples. In some cases, the uncomplexed metal was removed by passing the solution through a cation exchange resin. Dilute sodium hydroxide and hydrochloric acid were used to adjust the pH of the complex solutions from 1 to 14. The metal content of the complex was determined according to the methods described above.

Similarly, bifunctional chelate conjugates were prepared and tested at pH 2.8.4 and 6.0. The amount of conjugate remaining in the solution was determined by HPLC using a radiometric detector.

Example 10 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid lutetium (III) Compound Prepared according to Example 5 An aqueous solution of EA-DO3A was prepared at a final concentration of 0.01 M.

Prepare a solution of lutecium chloride by dissolving lutecium chloride in 0.1 N hydrochloric acid at 3.9 m / ml

EN 221 187 Β1 at a concentration corresponding to lutetium chloride (0.01 mol Lu). Lutecium 177 is used as a tracer.

μΐ 0.01 M Lu solution was mixed with 2 μΐ Lu177 solution, 30 μΐ ligand and HEPES buffer (0.1 M, pH 7.6) was added to make a total volume of 1 mL. The resulting solution has a concentration of 0.3 mmol for both the ligand and lutetium.

The amount of complexed lutetium as determined by the cation exchange method was 98%.

Example 11

- [2- (4-Amino-phenyl) -ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-tri-acetic acid-yttrium (III) complex; [Y (EA-DO3A)]

The 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid ligand prepared according to Example 5 is converted into a mixed protonated ammonium salt with a strong cation exchange resin treatment (SP-Sephadex ™ C-25, Pharmacia). The column in protonated form is washed with distilled water and then the concentrated aqueous ligand solution is discarded and washed again with distilled water. The ligand was eluted with 0.5 M NH ₄ OH. The eluate was evaporated to dryness using a rotary evaporator and dried in vacuo.

of Y (OAc) 34H ₂ solution (0.203 g, 338.101 g / mol, 0.600 mmol) in distilled water was mixed with a warm solution of protonated ammonium salt ligand (0.3 g, 499.615 g / mol, 0.6 mmol) in 10 mL of distilled water. ). The solution was then heated to reflux and adjusted to pH 7 with 0.1 M ammonium hydroxide. After heating under reflux for 15 minutes, the solution is cooled and evaporated to dryness using a rotary evaporator. Excess ammonium acetate was removed by heating in an oil bath at 105 ° C under vacuum. The title compound, containing 1 equivalent of ammonium acetate, was obtained in a yield of 379 mg (99%).

13 C-NMR (88 ° C, D 2 O, 75 MHz):

24.4, 28.4, 51.2, 56.5, 57.2 (2C), 57.7, 67.2, 67.6,

120.5.132.3.135.2, 182.1,182.5, 183.0.

FAB MS m / e 552 (positive ion, [M ⁺ H ⁺ ] ⁺ ), m / e 610 (negative ion, [M ⁺ OAc -] -).

Example 12 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid-samarium complex;

[Sm (EA -DO3A)]

In the same manner as in Example 11, Sm (OAc) 3 3 H ₂ (0.229 g, 381.531 g / mol, 0.6 mmol) was used in place of the yttrium compound. The title compound, containing 1 equivalent of ammonium acetate, was swallowed with 408 mg (99%).

13 C NMR (88 ° C, D 2 O, 75 MHz):

23.8, 27.9, 51.4, 57.5, 58.1 (3C), 68.7, 73.4, 120.3,

132.2, 134.7,185,0,186.8, 192.1;

FAB MS m / e 615 (positive ion, [M ⁺ H ⁺ ] ⁺ , Sm isotope), m / e 673 (negative ion, [M + OAe ^- ] *, Sm isotope).

Example 13 1- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid-samarium (III) complex 1.48 mg / 100 μΐ ( solution of the compound prepared in 0.03 M) concentrations of Example 8 was mixed with 10 μΐ 15 μΐ of n hydrochloric acid, and containing Sm at 153 SmCl ₃ -oldattal. The volume was then made up to 1 ml with 1 ml of NANOpure ™ water, and the solution was divided into two 500 μΐ fractions and one was heated at 100 ° C for 1 hour. The metal in the complex is then determined in both the cooled and non-cooled samples by the known cation exchange method. The results show that the complex contains 81% and 43% of the metal in the heat-treated and non-heat-treated samples, respectively.

Example 14 Stability of 1- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid-samarium (UI) complex as a function of pH

The heat treated sample of Example 13 was divided into 2 aliquots. The pH of one aliquot is adjusted with hydrochloric acid and the other with sodium hydroxide according to the following table. After reaching the desired pH, the samples are allowed to stand for another 10 minutes and the metal content of the complexes is determined by a known cation exchange method. The results are summarized in the following table.

pH Complex, % 1 20 2 37 4 49 5 58 7 81 9 62 10 64 12 64 13 54

Example 75 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid samarium (III) Complex 4.65 mg / 100 A solution of 1- [2- (4-aminophenyl) ethyl] 1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid obtained in Example 8 was prepared in a concentration of 3 ml. -t SmCl ₃ -oldattal mixed with 15 μΐ 0.1N Sm-153-t. The volume was then made up to 1 ml with NANOpure ™ and the pH was adjusted to 7.3 with sodium hydroxide. The solution was then divided into 2 aliquots, one of which was heated at 100 ° C for 1 hour. The metal content of the complexes is then determined in both heat-treated and non-heat-treated samples by the known cation exchange method. The results obtained showed 96% and 93% metal content of the heat-treated and non-heat-treated samples, respectively.

HU 221 187 Β1

Example 16

- Stability of [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecem-4,7,10-triacetic acid samarium (III) as a function of pH

The heat treated and untreated samples of Example 15 were divided into two aliquots. One aliquot is adjusted to pH with hydrochloric acid and the other with sodium hydroxide in the following table. After reaching the desired pH, the samples were allowed to stand for a further 10 minutes and then the metal content of the complexes was determined by a known cation exchange method. The results are summarized in the following table.

Complex, % PH heat treated Not heat-treated 1 44 13 3 50 42 5 73 55 7 96 93 9 98 98 11 89 90 13 28 37

Biological examples

Bifunctional chelates containing a metal chelating group such as DTPA and a reactive linker (arylamine) within the molecule are known to be capable of covalently bonding to various targeted target biomolecules specific for cancer or tumor cell epitopes or antigens. Radioactive nucleus complexes of such conjugates can also be used for diagnostic and / or therapeutic purposes because they are capable of delivering a radioactive nucleus to a cancerous or cancerous cell [Meares et al., Anal. Biochem. 142: 68-78 (1984); see also pp. 215-216. J. Protein Chem., 3 (2) (1984), Meares and Goodwin, and U.S. Patent No. 4,678,677, issued July 7, 1987 to Warshawsky et al. March 24, 1987, Brechbiel et al., Inorg Chem., 25, 2772-2781 (1986). It is expected that the product containing the radioactive or luminescent metal may also be used in in vitro immunoassays.

Prior art information

The applicability of labeled antibodies depends on several different factors. For example: 1. the specificity of the antibody, 2. the integrity or stability of the complex under the conditions of use (i.e., serum stability), and 3. the integrity of the antibody, that is, that the specificity and immunoreactivity of the antibody are not affected.

The stability or integrity of the complexes is of particular importance when using the antibody conjugate containing the radioactive core as a diagnostic and / or therapeutic agent. For example, kinetic instability can lead to instability in the serum and cause dissociation of the radioactive nucleus from the complex. As such, it cannot be effectively used for diagnostic and therapeutic purposes. In addition, radiation damage to normal cells is greater in this case [Colé et al., J. Nucl. Med., 28, 83-90 (1987)].

Binding of the Radioactive Seed to the Antibody The radioactive nucleus is bound to the antibody by either binding the BFC to the antibody in a known manner and then chelating the radioactive nucleus under conditions compatible with the antibody or otherwise by metal BFC to the antibody. curing the complex at ambient or elevated temperatures. [Meares et al., Acc. Chem. Res., 17, 202-209 (1984)]. The following examples illustrate this.

Example 7A (Comparative Example) Binding of 1- (4-Isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid-samarium-153 complex to IgG or the F (ab) 2 fragment of CC-49; [ ^IS3 Sm (SCN-Bz-DTPA)] IgG and [ ^{, 53} Sm (SCN-Bz-DTPA)] - F (ab) 2 fragment 1- (4-Isothiocyanato-benzyl) -diethylene-triamine-pentaacetic acid-samarium- 153 complexes ( ¹⁵³ Sm (SCN-Bz-DTPA)) are prepared by mixing 150 µl of ¹⁵³ Sm in 0.1N hydrochloric acid with 9 µl of 1- (4-isothiocyanato-benzyl) -diethylenetriaminepentaacetic acid HEPES buffer (0.5 mol, pH 8.9, about 30) was added to adjust the pH to 6. The resulting ¹⁵³ Sm complex is then mixed with 22.5 x 10 ~ ⁹ moles of IgG, or CC-49 F (ab ') 2 fragment (1 x 10 ⁴ moles of protein in 50 moles HEPES buffer, pH 8.5), and adjust the pH to 8.9 with sodium carbonate solution (1 mol, 12-15 Si). The conjugation reaction was carried out at room temperature (about 25 ° C) for about 3 hours. The resulting IgG or F (ab ') 2 conjugate, which is labeled with the ^153S Sm complex, is separated and lysed in the II. Example 4 is tested and characterized.

Example I 1- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-tri-acetic acid-samarium-153 complex; [ ^{. 53} Sm (SCN-EA-DO3A)] complex To 100 µl of ¹⁵³ Sm / O, IN hydrochloric acid was added 5 µl of SCN-EADO3A (50 mmol in HEPES buffer, pH 8.2, prepared as in Example 6). followed by the gradual addition of 0.5 M HEPES buffer (pH 8.9) (about 25 µl) to adjust the pH to about 7. The process of chelation formation is detected by HPLC (GF-250 column, HPLC-System I). The yield is about 50%.

II. Example 1- 1- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid-samarium153 Complex Antibody ^Conjugate [ ^I53 Sm (SCN-EADO3A)] - IgG Conjugate

CC-49 (rat monoclonal IgG) was used as an antibody to the TAG-72 epitope (

EN 221 187 Β1 single antigen). To prepare the conjugate, 187 µl of antibody solution (1.2 x 1 (h in ⁴ moles of 50 ml HEPES, pH 8.5) was mixed with 4.5 x 10 ⁸ moles of ¹⁵³ Sm (SCN-EA-DO3A) complex as described in Example I. 1 M sodium carbonate solution was added to adjust the pH to 8.9 The reaction was carried out at room temperature for 2 hours, followed by IgG ¹⁵³ Sm by centrifugal gel filtration on Sephadex G-25 ( 2.2 ml) was separated on a disposable column and further purified by HPLC on a GF250 column, eluting with citrate buffer (0.25 mol, pH 7.4) The fractions containing the labeled IgG were collected, concentrated, three times in PBS. The resulting ¹⁵³ Sm-labeled IgG had a specific activity of about 0.16 pCi / pg Protein [ ¹⁵³ Sm (EA-DO3A)] IgG was intensified by HPLC [Sivakoff, SI, BioChrom. 1 (1): 42-48 (1986). ] as well as by standard biochemical methods such as sodium dodecyl sulfate: poly (acrylamide) gel electrophoresis and autoradiography, and solid phase radioimmunoassay (RIA) (see David Colcheer et al., Cancer Res. 43, 736-742). 1983)].

III. Example 1- Conjugate of 1- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid-samarium153 complex CC-49-F (ab ') 2 fragment; [ ⁱ³³ Sm (SCN-EADO3A)] - Fragment of CC-49 F (ab ') 2 CC-49 F (ab') 2 [produced by Lamoyi and

Nisonoff, A. by enzymatic digestion, E. Lamoyi, and A. Nisonoff, J. Immunol. Methods, 56, 235-243 (1983)] (225 µl of a 1 x 10 ~ ⁴ molar solution in 50 ml of HEPES buffer, pH 8.5) was mixed with 2.9 x 10 ⁸ mol of ¹⁵³ Sm (SCN-EA-DO3A) complex prepared according to Example I. To the resulting mixture was then added about 5 pmoles of sodium carbonate to adjust the pH to 8.9 and the reaction was continued for 2.5 hours. The resulting ¹⁵³ Sm fragment is separated and the II fragment is isolated. Example 4a.

ARC. Examples (A and B) [ ^J53 Sm (SCN-EA-DO3A)] - IgG and [ ^'33 Sm (SCN-EADO3A)] - In vivo Localization of F (ab) 2 A ¹⁵³ Sm (SCN-EA-DO3A) - IgG (Example II) and -CC-49 F (ab ') 2 fragment conjugate (Example III) were characterized by uptake of labeled substances in human tumor-infected athymic mice. Female athymic mice (Nu / Nu, CDI background) were inoculated (SC) subcutaneously (0.1 mL / source) with the human colon carcinoma cell line, LS-174T (approximately 1 x 10 ⁶ cells / animal). About 2 weeks after inoculation, each animal was injected via the tail vein with about 2 pCi (15-30 pg) of ¹⁵³ Sm antibody in PBS. The mice were then sacrificed at various time intervals, transfused, and the tumor as well as the selected tissues excised and weighed, and radioactivity was determined using a gamma counter. The number of strokes per minute (CPM) for ¹⁵³ Sm was determined for each tissue and expressed as CPM per gram of tissue per injected dose x 100 (percentage of injected dose / g). The results obtained are shown in Figures 1-14. 1A and 1A. and IB. are summarized in tables. For comparison, ¹⁵³ Sm (SCN-BzDTPA) IgG and -CC-49 F (ab ') 2 fragment conjugates (Example ZA) were used. Their results are reported in IC. and ID. tables.

The following notations have been used in the figures and in the following tables:

conjugates Signal Spreadsheet Figures Biological example [ ¹⁵³ Sm (SCN-EA-DO3A)] - IgG-CC-49 Δ IA. 1-7 II [ ¹⁵³ Sm (SCN-EA-DO3A)] - F (ab ') 2-CC-49 Δ IB. 8-14 III [ ¹⁵³ Sm (SCN-Bz-DTPA)] - IgG-CC-49 SHE IC. 1-7 ZA [ ¹⁵³ Sm (SCN-Bz-DTPA)] - F (ab ') 2-CC-49 SHE ID. 8-14 ZA

In the figures attached to the description, the data are as follows:

Figure Organ Title First Blood ¹⁵³ Sm-BFC * biodistribution Second Liver ^l53 Sm-BFC * biodistribution Third spleen ^l53 Sm-BFC * biodistribution 4th Kidney ¹⁵³ Sm-BFC * biodistribution 5th Tumor ¹⁵³ Sm-BFC * biodistribution 6th thigh bone ¹⁵³ Sm-BFC * biodistribution 7th Total body retention ¹⁵³ Sm-BFC * biodistribution

Figure Organ Title 8th Blood ^l53 Sm-BFC * biodistribution 9th Liver ¹⁵³ Sm-BFC * biodistribution 10th spleen ¹⁵³ Sm-BFC * biodistribution 11th Kidney ¹⁵³ Sm-BFC * biodistribution 12th Tumor ¹⁵³ Sm-BFC * biodistribution 13th thigh bone ^1S3 Sm-BFC * biodistribution 14th All ¹⁵³ Sm-BFC * biodistribution

* To CC49 in mice with G LS 174-T tumor ** In mice with CC40-F (ab ') 2 LS174-T tumor

HU 221 187 Β1

IA. Table ¹⁵³ [ ¹⁵³ Sm (SCN-EA-DO3A)] - Biodistribution of ¹⁵³ Sm injected as IgG-CC-49 inj. dose / gram% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 23.08 2.36 17.25 2.40 11.32 * 1.06 5.78 * 1.69 Liver 6.95 1.28 6.58 0.56 6.58 0.62 7.62 0.98 spleen 5.17 0.59 5.06 0.96 4.62 0.72 4.92 ^'72 Kidney 4.59 0.68 4.01 0.53 3.74 0.76 3.20 0.59 Tumor 11.44 4.13 27.19 2.12 53.01 9.10 59.01 13.23 thigh bone - - 2.58 0.52 2.78 0.20 4.22 0.17 Tumor weight / g 0.16 0.09 0.20 0.09 0.30 0.17 0.52 0.34

* n = 4

IB. [ ¹⁵³ Sm (SCN-EA-DO3A)] - Distribution of ¹⁵³ Sm injected with F (ab ') ₂ CC-49 inj. dose / gram% (n = 5)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 13.36 0.97 1.52 0.42 0.20 0.06 0.05 0.01 Liver 7.13 0.65 8.59 1.3 8.08 0.8 8.11 0.92 spleen 5.94 1.24 4.96 1.27 4.81 0.99 5.08 1.43 Kidney 41.60 5.60 64.32 7.88 64.12 12.59 * 37.63 6.57 Tumor 12.95 1.73 16.84 3.71 11.50 4.56 5.53 1.46 thigh bone 2.74 0.65 2.45 0.26 3.41 0.75 4.99 0.29 Tumor weight / g 0.25 0.20 0.34 0.25 0.28 0.14 0.44 0.27

* N = 4

IC. Table 15 [ ¹⁵⁵³ Sm (SCN-Bz-DTPA)] - Bioavailability of ¹⁵³ Sm injected as IgG-CC-49 inj. dose / gram% (n = 10)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 24.97 3.07 16.05 2.45 12.81 2.69 5.84 1.76 Liver 7.74 1.35 6.01 0.95 5.68 1.07 5.43 1.45 spleen 5.69 1.04 4.86 1.36 4.72 0.81 3.78 0.63 Kidney 4.52 1.04 3.96 0.88 3.81 0.61 3.76 0.31 Tumor 13.90 3.18 42.86 14.03 46.33 8.30 61.32 19.80 thigh bone 2.36 0.12 1.95 0.38 1.97 0.75 2.25 0.50 Total body retention 85.45 4.74 80.16 3.58 77.36 5.32 70.60 3.32

HU 221 187 Β1

ID. Table ¹⁵³ [ ¹⁵³ Sm (SCN-Bz-DTPA)] - Bioavailability of ¹⁵³ Sm injected as F (ab ') ₂ -CC-49 inj. dose / gram% (n = 10)

Organ 5 hours 24 hours 48 hours 120 hours AVG STD AVG STD AVG STD AVG STD Blood 15.03 1.84 2.13 0.48 0.29 0.08 0.04 0.02 Liver 7.07 0.88 7.03 1.24 6.14 0.67 5.80 1.00 spleen 4.50 0.97 4.22 1.15 3.65 0.62 3.24 0.42 Kidney 46.74 8.69 80 12.76 61.42 9.85 30.17 5.17 Tumor 15.95 3.88 18.66 5.10 15.55 2.80 6.54 1.14 thigh bone 2.48 0.24 1.77 0.28 2.48 0.40 2.85 0.35 Total body retention 83.23 4.64 72.17 6.20 58.89 4.40 40.53 2.48

Embodiments other than the preceding examples, which will be apparent to those skilled in the art, are also within the scope of the invention. The examples provided are merely illustrative of the invention and are not intended to limit the claims in any way.

Claims (22)

PATENT CLAIMS A process for the preparation of a compound of formula (III) and pharmaceutically acceptable salts thereof, wherein Q is CH ₂ -CO ₂ R, wherein R is hydrogen or C 1-4 alkyl, n is a number from 0 to 4, R ² is a nitro, amino or isothiocyanate group, characterized in that (a) a compound of formula (III '): wherein: R ² and n are as defined above, by reacting a compound for introduction of a -CH ₂ -CO ₂ R group, wherein R is as defined above, or (b) to form a compound of formula (III) wherein R ^{2 is} amino; reducing a suitable compound of formula (III) wherein R ² is nitro, or (c) preparing a compound of formula (III) wherein R ² = isothiocyanate, a corresponding compound of formula (III) wherein R ² is amino reacting with thiophosgene, or (d) hydrolyzing a corresponding compound of formula (III) wherein R = hydrogen, wherein R is hydrogen, and optionally any resulting compound into a pharmaceutically acceptable salt thereof. converted. 2. A process according to claim 1 for the preparation of the pharmaceutically acceptable salts of the compounds of formula (III) with potassium, sodium, lithium, ammonium, calcium, magnesium or hydrochloride salts, wherein the appropriate starting materials are used. 3. A process according to claim 1 for the preparation of a compound of formula III wherein R is hydrogen, the other substituents being as defined in claim 1, wherein the appropriate starting materials are used. The process according to claim 1, which is 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid-4,7,10-trimethyl. -ester ester compound, characterized in that the appropriate starting materials are used. The process according to claim 1 for the preparation of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid, characterized in that starting materials are used. The process according to claim 1 for the preparation of 1- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid, wherein the corresponding starting materials are used. The process according to claim 1 for the preparation of the hydrochloride salt of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid, characterized in that: using the appropriate starting materials. A process for use as a ligand of a compound of formula (III) or a pharmaceutically acceptable salt thereof Q is CH ₂ -CO ₂ R where R is hydrogen or C 1-4 alkyl, n is a number from 0 to 4, R ² is a nitro, amino or isothiocyanate group and for the preparation of complexes containing one of the following metal ions: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y or Se, characterized in that said compound of formula (III) or a pharmaceutically acceptable salt thereof is complexed with said metal ion. The process for the preparation of complexes according to claim 8, which comprises Sm, Lu or Y as metal ions, characterized in that the appropriate starting materials are used. HU 221 187 Β1 The process for the preparation of complexes according to claim 8, which comprises, as metal ^ions , ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁴⁴⁹ Pm, ¹⁵⁹ Gd, ¹⁴⁰ La, ^'77 Lu, ¹⁷⁵ Yb, ⁴⁷ Sc or ¹⁴² Pr. using the appropriate starting materials. The process for the preparation of complexes according to claim 9, comprising the following metal ions: ¹⁵³ Sm, ¹⁷⁷ Lu or ⁹⁰ Y, characterized in that the appropriate starting materials are used. The process for the preparation of complexes according to claim 10 or 11 which is a ligand according to claims 4-7. A compound of formula (III) according to any one of claims 1 to 3, characterized in that the appropriate starting materials are used. 13. A process for the preparation of a ligand of a compound of formula (III) Q is CH ₂ -CO ₂ R where R is hydrogen or C1-C4 alkyl, n is a number from 0 to 4, R ² is an amino or isothiocyanate group, one of the following metal ions: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y or Se and the antibody or antibody fragment Conjugates comprising: (a) covalently linking a compound of formula (III) or a pharmaceutically acceptable salt thereof in complexed form with said metal ion to an antibody or antibody fragment, or b) covalently conjugating a compound of formula III or a pharmaceutically acceptable salt thereof with an antibody or antibody fragment and complexing with a metal ion as above. 14. The method of claim 13 for preparing a conjugate, comprising one of the metal ion is ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹ 'Y, ¹⁴⁹ Pm, ¹⁵⁹ Gd,' ⁴ ° La, ¹⁷⁷ Lu, ¹⁷⁵ Yb, ⁴⁷ Sc or ¹⁴² Pr , characterized in that the appropriate starting materials are used. The process for the preparation of conjugates according to claim 14, which comprises ¹⁵³ Sm, ⁹⁰ Y or ¹⁷⁷ Lu per metal ion, characterized in that the appropriate starting materials are used. 16. A process for the preparation of conjugates according to any one of claims 1 to 5, comprising the monoclonal antibody or fragment thereof as an antibody or antibody fragment, characterized in that the appropriate starting materials are used. The method of making conjugates according to claim 16, which comprises as an antibody or antibody fragment: CC49 (ATCC HB 9459), CC-49 F (ab ') ₂ , CC-83 (ATCC HB 9453), CC-83 F. (ab ') ₂ or B72.3 (ATCC HB 8108), wherein the appropriate starting materials are used. 18. A process for the preparation of conjugates as claimed in any one of claims 4 to 7, which as ligand of claims 4-7. A ligand according to any one of claims 1 to 3, characterized in that the appropriate starting materials are used. 19. A process for the preparation of a pharmaceutical composition comprising the steps of 8 to 12. A complex prepared according to any one of claims 1 to 6, admixed with a pharmaceutically acceptable carrier and converted to a pharmaceutical composition for administration. 20. A process for the preparation of a pharmaceutical composition comprising the steps of any one of claims 13-18. The conjugate produced by the process of any one of claims 1 to 6 is mixed with a pharmaceutically acceptable carrier and converted into a pharmaceutical composition for administration. 21. A diagnostic composition, characterized in that a diagnostic composition according to any one of claims 8-12. A complex prepared according to any one of claims 1 to 6 in admixture with a pharmaceutically acceptable carrier. 22. A diagnostic composition, wherein the diagnostic composition of any one of claims 13-17. Conjugate prepared according to any one of claims 1 to 6 in admixture with a pharmaceutically acceptable carrier.