FI104898B - Process for the preparation of a therapeutically active metal complex compound - Google Patents

Description

, 104898

A process for preparing a therapeutically active metal complex compound

The present invention relates to a process for the preparation of a therapeutically active metal complex compound represented by the formula (IA)

Q

/ "3 10, -ζ V \ / R2 - // \\ - CCH2) -c-N K-a> = /";

Q

A compound according to 15 wherein Q is independently hydrogen or (CHR 5) p CO 2 R, Q 1 is hydrogen or CO 2 R, each R is independently hydrogen or C 1 -C 4 alkyl, provided that at least two of Q and Q 1 are other than hydrogen and that when Q1 is hydrogen then R3 is other than hydrogen, each R5 is independently hydrogen or C1-4 alkyl, n is an integer from zero to 5, p is 1 or 2, R2 is hydrogen or nitro, amino, isothiocyanato , semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl groups, R 3 is C 1-4 alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen, R 4 is hydrogen or nitro, amino, isothiocyanato, semicarboxyl; a bazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl group, provided that R 2 and R 4 cannot both be hydrogen but one must be hydrogen, 2 104898 or a pharmaceutically acceptable salt thereof, complexed with a metal ion La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y or Se.

Functional group-containing chelating agents, or bifunctional coordinating compounds, are known to be capable of covalently binding to an antibody specific for epitopes or antigens of cancer or tumor cells. Radionuclide complexes 10 of such antibody-chelating agent conjugates are useful in diagnostic and / or therapeutic applications as a means of transporting the radionuclide to a cancer or tumor cell. See, for example, Meares et al., Anal. Biochem. 142: 68-78 (1984) and Krejcarek et al., Biochem. and Biophys. Res. Comm. 77: 581-15155 (1977).

Chelate-forming aminocarboxylic acids have been known and studied for many years. Typical aminocarboxylic acids include nitrile acetic acid (NTA), ethylenediamine tetraacetic acid (EDTA), hydroxyethylethylenedioxy-20-amine triacetic acid (HEDTA), diethylenetriamine pentacetic acid (DTPA), trans-1,2-diaminocarboxy-10 -tetra-azacyclododecane-tetraacetic acid (DOTA). Numerous aminocarboxylic acid-based bifunctional chelating agents are disclosed; '' 25 ty and made. For example, cyclic dianhydride of DTPA has been reported [Hnatowich et al., Science 220: 613-615 (1983); U.S. Patent 4,479,930] and DTPA Carboxylic Acid Mixed Anhydrides [Gansow, U.S. Patent Nos. 4,454,106 and 4,472,509; Krejcarek et al., Biochem. 30 and Biophys. Res. Comm. 77: 581-585 (1977)]. When the anhydrides are coupled to the proteins, attachment occurs via the formation of an amide bond, leaving four of the original five carboxymethyl groups of the diethylenetriamine backbone (DETA backbone) [Hnatowich et al., Int. 35 J. Appl. Large. 33: 327-332 (1982)]. U.S. Patent Nos. 3,104,898, 4,432,907 and 4,352,751, further disclose bifunctional chelating agents which are suitable for binding metal ions to "organic species, such as organic target molecules or antibodies".

As above, the coupling is effected via the amide group utilizing the dianhydrides of diaminotetraacetic acids. Examples of anhydrides are dianhydrides of EDTA, CDTA, propylene diamine tetraacetic acid and phenylene 1,2-diamine tetraacetic acid. Recent U.S. Patent 10,464,447,437 discloses a plurality of complex salts formed from the anion of a complexing acid for use in various diagnostic techniques. Conjugation is mentioned to occur via the carboxyl group of the complex forming acid, which results in binding via an amide-15 bond.

Paik et al. in J. of Radioanalytical Chemistry 57 (1980), No. 12, 553-564 have disclosed the use of p-nitrobenzyl bromide in the reaction with "protected" diethylene triamine, i.e. bis (2-phthalimidoethyl) amine-20, followed by deprotection and carboxymethylation with chloroacetic acid to give N'-p-nitrobenzyldiethylenetriamine-Ν, Ν, Ν '', N'1-tetraacetic acid. Since the bonding is via nitrogen, tetra-; 25 acetic acid derivative. The disclosure relates to conjugation and chelation of a bifunctional chelator with indium. Substitution on the nitrogen atom is also described by Eckelman et al. in J. of Pharm. Sci. 64 (1975), No. 4, 704-706, which substitution occurs by reacting amines such as "ethylenediamine or diethylenetriamine with the appropriate alkyl bromide before carboxymethylation". Compounds have been proposed as potential agents for use in radiopharmaceutical imaging.

4, 104898

Another group of bifunctional chelating agents based on the amino carboxylic acid functionality is well documented in the literature. Sundberg, Meares et al. in J. of Med. Chem. 17 (1974), 5, No. 12, 1304, discloses bifunctional analogs of EDTA. Typical examples of these compounds are 1- (p-aminophenyl) ethylenediaminetetraacetic acid and 1- (p-benzenediazonium) ethylenediaminetetraacetic acid. The publication discusses the attachment to proteins via the para-10 substituent and the binding of radioactive metals to the chelating group. The compounds are also disclosed in Biochemical and Biophysical Research Communications 75 (1977), No. 1, 149, and in U.S. Patent Nos. 3,994,966 and 4,043,998. It is important to note that the binding of an aromatic group to the EDTA structure occurs at the carbon atom of the ethylenediamine backbone. through. Optically active bifunctional chelating agents based on EDTA, HEDTA, or DTPA are disclosed in U.S. Patent No. 4,622,420. In these compounds, the 20 alkylene group combines an aromatic group (containing a functional group required for protein attachment) with a chelate-containing functional group. Other sources treating such compounds include Brechbiel et al., Inorg. Chem.

: 25 25: 2772-2781 (1986), U.S. Patent 4,647,447 and International Patent Publication No. WO 86/06384.

More recently, U.S. Patent No. 4,678,667 and Moi et al., Inorg. Chem. 26, 3458-3463 (1987) disclose some macrocyclic bi-functional chelating agents and their use of copper sulfate / plate conjugates for diagnostic or therapeutic applications. The attachment of the functional aminocarboxylic acid moiety to the rest of the bifunctional chelating agent and molecule occurs via the ring carbon atom of the cyclic polyamine ring-35 gon. The linking group, which is 5 1G4898 - at one end bound to the ring carbon of a cyclic polyamine, is also bound at one end to a functional group capable of reacting with the protein.

Another group of noteworthy bifunctional chelating agents are compounds in which the chelating moiety of the molecule, i. aminocarboxylic acid, is attached via nitrogen to a functional group of a molecule containing a reactive moiety with protein 10. For example, Mikola et al. in their patent application (International Publication No. WO 84/03698, published 27.9.1984), a bifunctional chelating agent prepared by reacting p-nitrobenzyl bromide with DETA followed by reaction with bromoacetic acid to give the aminocarboxylic acid. The nitro group is reduced to the corresponding amine group and then converted by reaction with thiophosgene to the isothiocyanate group. These compounds are bifunctional chelating agents capable of forming chelates with lanthanides that can be conjugated to bio-organic molecules for use as diagnostic agents. Because the binding of the linker moiety of the molecule occurs via the nitrogen of one of the aminocarboxylic acids, one potential coil is lost; 25 nascent aminocarboxyl group. Thus, a DETA-based bifunctional chelator having four (rather than five) acid groups is prepared. In this regard, the chelating agent belonging to this group is similar to those which are attached to the protein via an amide group, resulting in the loss of the chelating carboxyl group.

Carney, Rogers, and Johnson recently presented at the 3rd International Conference on Monoclonal Antibodies for Cancer, San Diego, California, June 4, 6, 1988, 35 abstracts entitled "Absence of Intrinsically« 104898

Higher Tissue Uptake from Indium-111 Labeled Antibodies: Co-Administration of Indium-111 and Iodine-125 Labeled B72.3 in a Nude Mouse Model and Influence of Chelator Denticity on the Biodistribution of Indium-Ill Labeled 5 B72.3 Immunoconjugates in Nude Mice ". They reveal the biodistribution of indium-111 complexed with the bi-functional EDTA and DTPA chelator. Binding of the aromatic ring to the EDTA or DTPA moiety occurs via acetate methylene. In a recent meeting (Florida Conf. on Chem. In Biotechnology, April 26-29, 1988} Bifunctional derivatives of EDTA and DTPA in which the p-isothiocyanate benzyl group is bound to a methylene carbon of a carboxymethyl group have also been disclosed by DK Johnson et al. U.S. Patent Nos. 4,088,747 and 4,091,088 (1978) to ethylene diamine acetic acid (EDDA) -chelated chelating agents with EDDA aromatic ring incorporation. is carried out via alkylene or acetate methylene. The compounds are mentioned as being useful as chelates in the study of pancreatic and biliary function. The preferred metal is technetium-99m. Indium-111 and indium-113m are also mentioned as suitable radionuclides for the description.

Thus, it would be advantageous to provide a complex that is not readily degraded, rapidly eliminated throughout the body except the desired tissue, and conjugates with the antibody to achieve the desired results.

The figures shown in the accompanying drawings can be illustrated as follows: Figures 1-7 and 15-21 show the biological distribution of 153Sm given as a 153Sm-containing conjugate prepared from the metal complex compound of this invention. The conjugate in question used the antibody CC49-IgG. Biodistribution was determined in nude mice bearing LS 174-T tumor.

104898

Figures 8-14 and 22-28 show the biodistribution of 153Sm given as a 153Sm conjugate of the metal complex compound obtained according to the present invention. In this conjugate, CC49-F (ab ') 2 was used as 5 antibody fragments. Biodistribution was determined in nude mice bearing LS 174-T tumor.

Figures 29-34 show the bio-10 distribution of 177Lu given as a conjugate containing 177Lu (PA-DOTMA) or 177Lu (PA-DOTA). The conjugate used was CC49-IgG antibody. Biodistribution was determined in balb / C mice bearing LS 174-T tumor.

Surprisingly, the complexes obtained in accordance with the present invention are relatively stable (i.e., not easily digestible) and some of them are rapidly eliminated throughout the body and in some non-target organs such as liver, kidney, and bone.

The invention relates to the design and manufacture of novel bifunctional chelating agents, each containing a chelating functional group and a chemically reactive group for covalent binding to biomolecules. Related thereto are methods for the preparation of various bifunctional coordinating compounds (BFCs) and metal complexes, as well as conjugates containing the complex with antibodies for diagnostic and / or therapeutic uses, such as radionuclide 153: 11a, lutetium-177 or yttrium-90) to generate labeled antibodies and / or fragments.

In the present invention, the novel bifunctional chelating compounds of formula IA form complexes with metal ions, in particular with "radioactive" metal ions of the rare-earth type. Such rare earth metal ions are La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y and Se, and preferred are Sm, Ho, Y and Lu. Preferred radioactive ions of rare earths are 153Sm, 166Ho, 90Y, 149Pm, 5159Gd, 140La, 177Lu, 175Yb, 47Sc and 142Pr, and particularly preferred 153Sm, 166Ho, 90Y and 142Pr. Other radioactive metal ions that may be of interest are 47Sc, 99mTc, 186Re, 188Re. 97Ru, 10SRh, 109Pd, 197Pt, 67Cu, 198Au, 199Au, 67Ga, 68Ga, 1x1In, 113min, 115min, 117Sn and 212Pb / 212Bi. Thus, the complexes formed may be linked (covalently bonded) to the antibody or fragment thereof and used for therapeutic and / or diagnostic purposes. The complexes and / or conjugates may be formulated for in vivo or in vitro use. Formulated conjugates are preferably used in the treatment of cancers in animals, particularly humans.

The complexes obtained according to the present invention containing a non-radioactive metal can also be used for the diagnosis and / or treatment of disease states such as cancer. Such use is known for non-radioactive metals in the use of radio frequency for the induction of hyperthermia (JP Patent Specification Kokai Tokkyo Koho 61/158,931) and in fluorescence immuno-guided therapy (FIGS) [K. Pettersson et al., Clinical Chemistry 29 (1983), No. 1, 60-64 and C.

"" 25 Meares et al., Acc. Chem. Res. 17: 202-209 (1984)].

The term "mammal", as used herein, refers to animals that feed their offspring with milk secreted by the mammary glands, preferably even mammals, more preferably humans. By "antibody" is meant any polyclonal, monoclonal or chimeric / antibody or heterova antibody, preferably a monoclonal antibody; By "antibody fragment" is meant Fab fragments and (F (ab ') 2 fragments, as well as any portion of an antibody that is specific for the desired epitope or epitopes. As used herein, the term "metal chelate antibody conjugate" or " conjugate "moiety" refers to whole antibodies and / or antibody fragments, including their semi-synthetic or genetically engineered variants.

"Complex" as used herein means a compound of the formula IA of the invention complexed with a metal ion of a rare earth type, in particular with a radioactive rare earth ion, wherein at least one metal atom of 10 ml is chelated or complexed; "radioactive metal ion chelate antibody conjugate" or "radioactive metal ion conjugate" means a radioactive metal ion conjugate in which the metal ion is covalently bound to the antibody or antibody fragment; "radioactive" as used in the context of the term "metal ion" means one or more isotopes of elements of the rare earth type which emit particles and / or photons, such as 153Sm, 166Ho, 90Y, 20 149Pm, 159Gd, 140La, 177L, 177L. , 47Sc and 142Pr; the terms "bi-functional coordinating compound", "bifunctional chelating agent" and "functional group-containing chelating agent" are used interchangeably and refer to compounds containing a chelating moiety capable of forming a chelate with a metal ion, and a covalently bonded linker / spacer capable of acting as a vehicle for covalent binding to an antibody or antibody fragment.

"Pharmaceutically acceptable salt", as used herein, means any salt of a compound of formula IA that is sufficiently non-toxic to be useful in the diagnosis and treatment of mammals. Thus, salts may be used in accordance with the present invention. Typical salts such as those formed by conventional 104898 reactions from both organic and inorganic sources include, for example, sulfur, salt, phosphorus, vinegar, amber, lemon, milk, maleic, fumaric, palmitic, bile, pamoni, mucus, glutamine, D-camph-5 ri, glutar, glycol, phthalic, wine, ants, laurine, stearin, salicyl, methanesulfone, benzenesulfone salts of sorbic acid, sorbic acid, picric acid, benzoic acid and cinnamic acid and other suitable acids. Salts also include salts formed by conventional reactions from both organic and inorganic sources, such as ammonium ions, alkali metal ions, alkaline earth metal ions and the like. Particularly preferred are the potassium, sodium and ammonium salts of the compounds of formula IA and mixtures thereof.

Of course, the free acid form of the compounds of formula IA as well as the protonated form of the compounds, such as that resulting from the protonation of the carboxylate and / or nitrogen atoms, i.e. the HCl salt of the formation, can be used.

Preferred compounds of formula IA are those compounds wherein Q1 is hydrogen A and are represented by the following formula: - (-) - (-) - (CH2) -C-NN-

(II) Q

Wherein each Q is independently hydrogen or CHR 5 CO 2 R and each R is independently hydrogen or C 1-4 alkyl, provided that at least two of Q are non-hydrogen, m is an integer from zero to five, R 2 is hydrogen or nitro -, amino, isothiocyanato-semicarbat-5-bond, thiosemicarbazido, carboxyl, bromoacetamido or maleimido group, R 3 is C 1-4 alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen, R 4 is hydrogen or nitro, amino, isothiocyanato-, , semicarbazido, thiosemicarbazido, carboxyl, bromoacetamide-10-doo or maleimido, and each R 5 is independently hydrogen or C 1-4 alkyl, provided that R 2 and R 4 cannot both be hydrogen, but one of them must be hydrogen; and pharmaceutically acceptable salts thereof.

When R3 and R4 are both hydrogen in formula II, the compounds correspond to the following formula:

Q

V

R-2 - <CH> - 'c ^

(III) Q

Wherein each Q is independently hydrogen or CHR 5 CO 2 R and each R is independently hydrogen or C 1-6 alkyl, provided that at least two of the substituents' Q are other than hydrogen, m is an integer from zero to five, R 2 is nitro, amino, an isothiocyanato, a semicarbazido, a thiosemicarbazido, a carboxyl, a bromoacetamido, or a ma-35-imimido group, and 12 104898 each R 5 is independently hydrogen or C 1-6 alkyl; or are the corresponding pharmaceutically acceptable salts.

When R 2 in formula II, the hydrogen compounds are of formula 5 3 '

(/ -cch2) -c-H *> - Q

\ - / "* / \ io - H

(IV) Q

wherein each Q is independently hydrogen or CHR 5 CO 2 R and each R is independently hydrogen or C 1-4 alkyl, provided that at least two of Q are non-hydrogen, m is an integer from zero to five, R 3 is C 1-6 alkoxy, -OCH 2 CO 2 H or hydroxy, R 4 is nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, bromoacetamido or maleimido, and each R 5 is independently hydrogen or C 1-6 alkyl; or are the corresponding pharmaceutically acceptable salts. Other preferred compounds of formula IA are those wherein at least one Q is hydrogen and are of formula

Q

r-S Q1. .. r2 - (/ y- (CH2) -c-n n-q

H * / / H

35 Q

(V) 13 104898 wherein each Q is independently hydrogen or CHR 5 CO 2 R, Q 1 is hydrogen or CO 2 R and each R is independently hydrogen or C 1 -C 4 alkyl, provided that at least two of Q and Q 1 are other than hydrogen and one Q is hydrogen, m is an integer from zero to five, R 2 is hydrogen or nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimido or bromoacetamido, R 3 is C 1-6 alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen, R 4 is hydrogen or nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimido or bromoacetamido groups, and each R 5 is independently hydrogen or C 1-4 alkyl, provided that R 2 and R 4 both cannot be hydrogen but one must be hydrogen; and pharmaceutically acceptable salts thereof.

Other preferred compounds of formula IA are those wherein Q 1 is CO 2 R and are of the formula?

: 25 r2 - {/ V- <ch2> -c-9 N-Q

Q

(VI) wherein v x x / 30 30 each Q is independently hydrogen or CHR 5 CO 2 R and *> each R is independently hydrogen or C 1 -C 4 alkyl, provided that at least one Q is other than hydrogen, m is an integer from zero to 5; , 14 104898 R 2 is hydrogen or nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimido or bromoacetamido, R 3 is C 1-4 alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen, R 4 is hydrogen or nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimido or bromoacetamido groups and each R 5 is independently hydrogen or C 1-4 alkyl, provided that R 2 and R 4 may not both be hydrogen but one of them must be hydrogen; and pharmaceutically acceptable salts thereof.

Some preferred compounds of formula VI are compounds wherein R 3 and R 4 are both hydrogen and corresponding to formula

Q

15 i rN— /

K - r M - (CH 2) -C-N N-Q

20 H

Q

(VII) wherein each Q is independently hydrogen or CHR 5 CO 2 R and: each R is independently hydrogen or C 1-4 alkyl, provided that at least one Q is other than hydrogen, m is an integer from zero to five, R 2 is nitro, amino, , isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, bromoacetamido or maleimido and each R 5 is independently hydrogen or C 1-6 alkyl; and pharmaceutically acceptable salts thereof.

Other preferred compounds of formula VI are compounds wherein R 2 is hydrogen and corresponding to formula 104898 wherein q R 3 - '.

f, CO 2 R 7

5 (y- (CH 2) -c-N N-Q

Q

(VIII) 10 each Q is independently hydrogen or CHR 5 CO 2 R and each R is independently hydrogen or C 1-4 alkyl, provided that at least one Q is other than hydrogen, m is an integer from zero to five, R 3 is C 1-4 alkoxy, - OCH 2 CO 2 H or hydroxy, R 4 is nitro, amino, isothiocyanato, semicarbazido, thiosemicarbazido, carboxyl, maleimido or bromoacetamido, and each R 5 is independently hydrogen or C 1-6 alkyl; And the pharmaceutically acceptable salts thereof.

The bifunctional chelating agent described herein (corresponding to any one of formulas IA to VIII) may be used to chelate or complex metal ions of the rare earth type, in particular; Radioactive rare earth ions to form metal ion chelates (also referred to herein as "complexes"). Since the functional moiety containing the complexes (represented by substituted phenyl in formula IA), they can be attached to functionalized supports, such as polymeric functional groups containing functional groups, or preferably in the case of a complex of formula IA wherein R 2 and R 4 are necessarily non-nitro groups, it can be linked to proteins or, more particularly, to antibodies or antibody fragments. The complexes described herein (represented by the rare earth metal ions, in particular the radioactive rare earth ions, the compound forming one of the formulas IA-VIII) may be covalently linked to antibody 5 or antibody fragment, and referred to herein as "conjugates".

Antibodies or antibody fragments useful in the conjugates described herein can be prepared by techniques known in the art. Highly specific mon-10 monoclonal antibodies can be prepared by hybridization techniques known in the art; see, for example, Kohler and Milstein [Nature 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976)]. Such antibodies usually have a very specific reactivity. Antibodies of any desired antigen or hapten may be used in the radioactive metal ion conjugates. Preferably, the antibodies used in the radioactive metal ion conjugates are monoclonal antibodies or fragments thereof having a high specificity for the desired epitope or epitopes. Antibodies used in this context may include, for example, antibodies against tumors, bacteria, fungi, viruses, parasites, mycoplasmas, differentiation and other cell membrane antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules. the enclosure. Some examples of antibodies or antibody fragments are C-11, CC-46, CC-49, F (ab ') 2 of CC-49, CC-83, F (ab') of CC-83. 2 and B72.3. [For antibodies to CC-49, CC-83 and B72.3, see D. Colcher et al., Cancer Res. 48 (August 15, 1988) 4597-4603. The Hybidomaoma cell line B72.3 is deposited in the American Type Culture Collection (ATCC) under the number HB 8108. Various CC antibodies are disclosed in U.S. Patent Application No. 7-35 073685. July 15, 1987 and is available through NTIS. Other murine monoclonal antibodies bind to epitopes on the TAG-72 antigen associated with tumors.] A more complete list of antigens can be found in U.S. Patent 4,193,983. The radioactive metal ion conjugates described herein are particularly advantageous for the diagnosis and treatment of various cancers.

Preferred complexes of rare-earth metals (lanthanides or pseudolanthanides) of the present invention are represented by Formula C [Ln (BFC)] (IX) wherein Ln is a rare-earth ion, such as Ce3 +, Pr3 *, Nd3 *, Pm3 *, Sm3 +, Eu3 *, Gd3 *, Tb3 *, Dy3 *, Ho3 *, Er3 *, Tm3 *, Yb3 * or Lu3 +, or a pseudolantanedione such as Sc3 +, Y3 * or La3 +, and particularly preferred metal ions are Y3 *, Ho3 +, Lu3 * and Sm3 *, BFC20 represents a bifunctional chelating agent of formula IA and C represents a pharmaceutically acceptable ion or ionic group having sufficient charge to render the entire complex electrically neutral. If the BFC contains at least 4 negatively charged groups, C is: a cation or a group of cations such as H *, Li +, Na *, K 1 R b *, C 5 \ Mg 2 *, Ca 2 *, Sr 2 *, Ba 2 *, NH 4 *, N (CH 3) 4 *, N (C 2 H 5) 4 *, N (C 3 H 7) 4 *, N (C 4 H 9) 4 *, As (C 6 H 5) 4 *, [(C 6 H 5) 3 P =] 2N * or other protonated amine . If the BFC contains 3 negatively charged groups, no C is required. If the BFC contains 2% of 30 negatively charged groups, C is an anion such as' 1 for example F ', Cl', Br ', I', C104 ', BF4', H2PO4, HC03 ', HCO2', CH3SO3, H3C-. C6H4-SO3-, PF6 ·, CH3CO2- or N (C6H5) 4 '.

The complexes obtained according to the present invention can be used in the form of formulations. The formulations si-35 contain a compound of formula IA combined with 104898 thallium ion and optionally an antibody and a suitable physiologically acceptable carrier, excipient or vehicle. Thus, the formulation may consist of a physiologically acceptable carrier and complex (metal ion + ligand) or conjugate (metal ion + ligand di + antibody). Methods for preparing such formulations are known. The formulations may be used in the form of a suspension, solution for injection or other suitable formulation. Physiologically acceptable suspending agents may be used, optionally in association with adjuvants.

An "effective amount" of the formulation is used for treatment. The dose will vary depending on the disease being treated. Although these formulations can be used for in vitro diagnosis, they are also possible for in vivo diagnosis. In addition, these formulations may also be used in radioimmuno-guided surgery (RIGS); however, other metals that could be used for this purpose include 99mTc, 311In, 113mIn, 67Ga and 68Ga.

Other uses of some of the complexes obtainable in accordance with the present invention include magnetic resonance imaging, for example, attachment of compounds of formula IA, in particular compounds of formula VI and Gd3 *, to polymeric carriers for various purposes, such as use as diagnostic agents, and lanthanide or removal of the pseudolanthanide metal ion by selective extraction.

The present invention provides complexes, some of which are more stable and / or have a better biodistribution and / or faster elimination from the body than are known in the art.

19 104898

The process of the invention for the preparation of a therapeutically active metal complex compound of formula IA from a compound or a salt thereof and a metal ion is characterized in that the compound of formula (A)

B

/ (VA)

10 o2fc - ('- (CH 2) -C-N M "H

n H

B

Wherein Q 1 is CO 2 R, R is C 1 -C 4 alkyl and n is an integer from zero to 5, is reacted with an α-halo carboxylic acid ester of the formula

BrCH2C02R

Wherein R is C 1 -C 4 alkyl, in the presence of an organic solvent and a base at a temperature between 0 ° C and reflux, to provide a compound of formula IA wherein Q 1 and n are as defined above, R 3, R 4 and R 5 are hydrogen, "R is C 1-4 alkyl and R 2 is nitro, or (B) the product of step (A) is reacted with a hydrazine such as Pd / C and hydrogen under standard reaction conditions for a compound of formula IA wherein Q 1 and n are as defined above, R 3, R 4 and R 5 are hydrogen, R 5 is C 1-4 alkyl and R 2 is amino, or (C) reacting the product of step (B) with a de-esterifying agent such as a strong acid with a compound of formula IA wherein Q1 and n are as defined above, R3, R4 and R5 are hydrogen, R is hydrogen and R2 is amino, or (D) reacting a compound of formula VA as defined above with an optically active α- benzene sulfonate of formula R5

) COOR

20 0

_ I

o - S = o ά. Wherein R and R 5 are as defined above, to provide a compound of formula IA wherein Q 1, R 5 and n are as defined above, R 3 and R 4 are hydrogen, R is C 1-4 alkyl and ·; R2 is nitro, or the product of step (D) of (E) is reacted with a Hydra reagent such as Pd / C and hydrogen under standard reaction conditions to provide a compound of formula IA wherein 104898 Q1, R5 and n are as defined above, R 3 and R 4 are hydrogen, R is C 1 -C 4 alkyl and R 2 is amino, or the product of step (E) of step 5 (F) is reacted with a de-esterifying agent such as a strong acid to provide a compound of formula IA Q 1, R 5 and n are as defined above, R 3, R 4 and R are hydrogen and R 2 is amino, or (G) a compound of the formula "/ R 1" / '- (CH 2) -C N N -h (IVA)

11 H

no2

B

Wherein R 3 'is C 1-4 alkoxy, -OCH 2 CO 2 H, or hydroxy and n is an integer from zero to five, is reacted with an α-halo carboxylic acid of the formula

BrCH2C02H

in the presence of an organic solvent and a base at a temperature between 0 ° C and 30 ° C to give a compound of formula IA wherein R 3 is as defined above R 3 ', n is as defined above, Q 1 and R 2 are hydrogen and R 4 is nitro, or 104898 (H) the product of step (G) is reacted with a Hydra reagent such as Pd / C and hydrogen under standard reaction conditions to provide a compound of formula IA wherein R 3 is as defined above for R 3 ', n is as defined above. as defined, Q 1, R, R 2 and R 5 are hydrogen and R 4 is amino, or (I) a compound of formula 10

B

/ OMe N ^ / - \ '1 \ / // v \ - (CH2) -C-M N' H (VIHA) 15 \ - / n '\

Λ-7 H

no 2 h where 2 0 Q 1 is CO 2 R, R is C 1-4 alkyl and n is an integer from zero to five, is reacted with an α-halo carboxylic acid derivative of the formula.

BrCH2C02R

wherein R is as defined above, in the presence of an organic solvent and a base at a temperature of 0 ° C and reflux temperature, to provide a compound of formula IA wherein Q 1, Q and n are as defined above, R 2 is hydrogen and R 4 is nitro, or 104898 (J) The product of step (I) is reacted with a de-esterifying agent such as a strong acid and then with a hydrogenating agent such as Pd / C and hydrogen under conventional reaction conditions to provide a compound of formula IA wherein Q1, Q and n are as defined above, R, R2 and R5 are hydrogen and R4 is amino, or the product of step (B), (F), (H) or (J) of (K) is reacted with thiophosgene to give the corresponding compound wherein R 2 or R 4 is isothiocyanato; (L) optionally reacting, optionally, a compound of formula IA with a suitable reagent to provide a potassium, sodium, lithium, ammonium, calcium, magnesium or hydrochloride adduct of the compound as a pharmaceutically acceptable salt, and (M) reacting a compound prepared by any of methods (A) to (L) with a metal ion selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, 20 Ho, Er, Tm, Yb, Lu, Y or Se.

One rational and common way to synthesize a macrocyclic, bifunctional chelating agent containing 12 ring atoms of formula IA is to monofunctionalize the free base macrocyclic compound (e.g.

.. 1,4,7,10-tetraazacyclododecane) by attaching only one of its nitrogen atoms to a suitable electrophile (e.g., an appropriately substituted α-halo carboxylic acid ester). This electrophile must contain a suitable linker group or a suitably protected linker group which enables the bifunctional ligand to be attached to the protein, antibody or antibody fragment.

It is known in the art that alkylation procedures for the preparation of mono-N-functional macrocyclic polyaza compounds may lead to mixtures of difficult to separate products [T. Hand, Top. Curr. Chem. 121: 24104898157-175 (1984)]. This problem has been overcome by using, as reported, large excesses of the macrocyclic compound (5 to 10 equivalents relative to the electrophile), which promotes the formation of the monoaddition product [M. Studer 5 and T. A. Käden, Helv. Chim. Acta 69: 2081-86 (1986); E. Kimura et al. , J. Chem. Soc. Chem. Commun. 1986, 1158-59].

Other pathways for the preparation of mono-N-functional macrocyclic polyaza compounds include an elongated protective, functionalization and deprotection system [P. S. Pallavicini et al., J. Amer. Chem. Soc. 109: 5139-44 (1987); M. F. Tweedle et al. , EP-A-0 232 751]. Abbott Laboratories has recently published a summary of a recent meeting report on the reductive amination of substituted 15-phenylphenolic acid and amines [D. K. Johnson et al., Florida Conf. is Chem. in Biotechnology, April 26-29, 1988, Palm Coast, FL].

General preparation routes for the preparation of chelating agents of formula IA are shown below in Synthetic Schemes I through V and include the reaction of a suitable electrophile with a macrocyclic polyaza compound in a suitable organic solvent at various reaction ratios, temperatures, and concentrations. Examples of suitable organic solvents include all hydrocarbons which promote solubility, such as acetonitrile, isopropanol, methylene chloride, toluene, chloroform, n-butanol, carbon tetrachloride, tetrahydrofuran and chloroform containing 5% ethanol, most preferably chloroform, n-butanol, 1,4-dioxane and acetonitrile. The macrocyclic compound and the electrophile can be used in stoichiometric or near stoichiometric amounts and the corresponding mono-N-alkylation product can be obtained in one step. The temperature in the reaction may range from about 35 ° C to about the reflux temperature of the mixture, and is 104898, preferably 0 to 25 ° C. The reaction time is about 1-24 hours.

A general procedure for the preparation of substituted α-haloic acid esters is desirable for the general use of Synthetic Cassettes II and IV. One suitable method is to brominate or chlorinate the acid halide formed in situ [e.g. D. N. Harpp et al., J. Org. Chem. 40 0: 3420-27 (1975)]. This procedure allows the exclusive alpha-halogenation of alkanoic acids even containing reactive benzyl groups. One general method for preparing substituted acid halides involves the reaction of an organic acid with thionyl chloride or sulfuryl chloride [e.g. Schwenk et al., J. Amer. Chem. Soc. 70, 3626-27 (1944)]. Both methods utilize free carboxylic acid, which is often available from commercial sources.

Macrocyclic polyaza compounds such as, for example, 1,4,7,10-tetraazacyclododecane may be prepared by methods already reported [e.g. Atkins T. J. et al., 20, 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-functional macrocyclic compound can be accomplished by the Desreux method using bromoacetic acid derivatives and a suitable base .. 25 [J. F. Desreux, Inorg. Chem. 19: 1319-24 (1980)].

All of the starting materials required in this invention are either commercially available or can be prepared from descriptions in the known literature.

In the following Scheme I, compounds of formula IA wherein Q1 is hydrogen are prepared. Although the terms mentioned refer to only one compound, other similar compounds of formula IA in which Q 1 is hydrogen and n is 35 to 5 may also be prepared by this method.

26 1 0 4 8 9 8

Scheme I

ΙΟΙ + Η "/ Ν'Η CHC1V 25 ° C Η-Η νο2 · '/' 10 / BrCH2C02CH3, / K2C03 CH3CN, / 25eC, 'l7 h o2n cooch3 15 00«,> —Ns /

Pd / c, H2, / ^ COOCH3 20 CH3OH, 25 ° C, / 2.5 h / h2h cooch3: · 25 ΥλλΙ ^ conc. HCl COOCH3 80eC, 2.5 h 7 N— '1 /

V

* ^ cooch3 / 30 * h7n cooh / _ / »» 35 ^ nHCl

XCOOH

27 104898

In the following Scheme II, compounds of formula IA wherein Q1 is hydrogen are prepared. Although the terms given refer to only one compound, other similar compounds of formula IA 5 wherein Q 1 is hydrogen and n is 0 to 5 may also be prepared by this method.

28 104898

Scheme II

B

NO 2 R 2 H

1) Pd / C, h2, ch3oh / 2) HCl (g), C2H5OH / 10 / H / joTUJ15 H2N h -nHCl

BrCH2CO2CH3, /

K 2 CO 3 and CH 3 CH

20 ^ cooch3 £ 0OCH3

1 ° fV

25 H2N ^ / cooch3 Väk. / HC1 / 30 / j / 0 ™ '· / cooh j @ X .. J' - h2n ^

35 COOH

29 104898

In the following Scheme III, compounds of formula IA wherein Q 1 is CO 2 R are prepared. Although the terms given refer to only one compound, other similar compounds of formula IA wherein Q 1 is CO 2 R and n is 0 to 5 may be prepared by this method.

30 104898

Scheme III Br _ ^ ~ C00H COOCH-, 5 ύ-a: .z: @

I CH 3 OH

02n no2

Br H

'V — COOCH,.

Ϋ '"CJ" ^

HO, 'H3COOC

1 H,

15 H

BrCH2CO2CH3,, K2CO3f CH3CN, \ 25eC, 17h \

Y

20 ON COOCH3 M / C, H2 Y / ~ n1 ^ _yCOOC £ 3 T H3C00 <:: · 25 HpN _ .COOCH, {ΊΘΐ C00CH> H ^ COOC \

l h2 ^ ___ \ / C00H

30 C00CH3 t ^ S00C 13HC1 k

COOH

104898

In the following Scheme IV, compounds of formula IA wherein Q 1 is CO 2 R are prepared. Although the terms given refer to only one compound, other similar compounds of formula IA wherein Q 1 is CO 2 R and n is 0 to 5 may be prepared by this method.

The A 'containing reagent shown in Scheme IV may be any leaving group suitable for generating the? -Acid, i.e., A' is phenyl or CF 3.

The use of an optically active alkylating agent has minimized the formation of diastereomers and simplifies the synthesis. It would be desirable to isolate a single diastereomer, which would produce one easily purified lanthanide complex, both for the formation of the radioactive chelate.

· •.

1 · 32 1 0 4 8 9 8

Scheme IV

H Br 'COOCH (CH 3) 2 5 »' C '+ if ^ l _,.

^ V% \ l \ QL

H N02 V / N N "H

(H3C) jCHOOC

10 "__ H

K 2 CO 3, CH 3 CH (50-95%)

H, C

3, COOCH-5

X

"R H

15 h3C ^, COOCH, O

o, 1 _ V I

2 V ^ v, H HlY _ 1] (H3C) 2CHOOC C „\ \ \ C00CH3 20 H I.jL · /> ch3 cooch3

Pd / C, H2 I

: · 25 ch3oh I

h3C <4 COOCH3 h3C <4 COOH

Β21νΛν H, V H2NV ^ N h 'V

. ^> ΓΊ ^ · ^> fl> (B3C) 2CHOOC 3 \ HOOC) \

.. COOCH3 COOH

H IIlL H uL

/ ▼ CH-j w conc. ECl »/ ▼ CH3

COOCH-, COOH

35 J

33 104898

In the following Scheme V, compounds of formula IA wherein Q 1 is CO 2 R and R 2 is hydrogen are prepared. Although the terms given refer to only two compounds, other similar compounds of formula IA wherein Q 1 is CO 2 R, n is 0 to 5, R 3 is as defined above, and R 2 and R 4 are present may be prepared by this method hydrogen and an amino, nitro, isothiocyanato, semicarbazido, thiosemicarbatido, maleimido, bromoacetamido and carboxyl group. 1 · 3 «104898

Scheme V

B

| R H OR '_ 1 [_ ΒΓ Xl

(ÄY "R * CH3 'i ^ YU ^' K w.H

+ H "^ Ks ^" H R * h 'dioxane kD> jJ

° 2N hr 02N

r'b h »R * CH3, 10 / R '= C0, CH, R1« H, COOCH3 R5 H ν' 2 3 ν 'BrCH2COOH BrCH2COOCH3

ν 'T

CH2COOCH3

15 "jC0" ca3o I

f V ~ K ~ ^ I t | 02l-H3

O) W ¥ ”J

chocooh CH2C00CH3

0, N

20 ° 2N 2

(1) HCl, 90-10 D ° C

(2) PtO 2 / H 2

PT02 / H2

▼ T

• · 9 c

: CH2C00H

CH2C00H

0H 1 CH 3 O I

i 1 / _X / N N-CH ^ COOH X \ 2 V '

f / Λγ / \ 2 xXx / -H N-CH-, COOH

(QJ (DJ

30 T c ^ coob 'h2cooh

E2S HjK

35 104898

The electrophilic moiety (the phenyl group substituted in Formula IA) may be prepared by methods known in the art. Such methods are disclosed in Acc. Chem. Res. 17, 202-209 (1984). One method for preparing compounds of formula IA wherein the electro-phenyl could be an isothiocyanate group (R2 or R4 present is an isocyanate group) comprises reaction of an aminocelate (R2 or R4 is an amino group) with thiophosgene and disclosed by MJ Fazion et al. U.S. Patent Application 289,712, filed December 23, 1988.

Radionuclides can be produced in many ways. The nuclide is bombarded in the nuclear reactor with neutrons to give a radionuclide, for example 15 Sm-152 + neutron - Sm-153 + gamma

Another method of providing radionuclides is to bombard the nuclides with particles in a linear accelerator or cyclotron. Another way is to isolate radionuclide 20 from a mixture of fission products. The method of generating radionuclides used in the present invention is not critical to the invention.

Conjugates may be prepared by first complexing and coupling with the invention, then an antibody or antibody fragment. Thus, the method comprises preparing the ligand, forming a complex with the metal, and subsequently coupling the antibody.

In the following examples, the following terms are used and the following conditions are used unless otherwise stated.

♦

Mass spectra were obtained either on a Finnigan TSQ mass spectrometer (Q 5 S model) or on a highly resolved VG ZAB-MS mass spectrometer (bombardment on fast x 10 1 8 4 9 9 8 atoms using a 3: 1 mixture of dithiothreitol and dithioerythritol).

1H and 13C NMR spectra were obtained using a Varian VXR-300, Bruker APC 300, IBM / Bruker Nr-80 or Jeol FX400 5 spectrometer. All spectra were obtained at 30 ° C unless otherwise noted. 1 H-NMR spectra were performed at 300 MHz, 80 MHz or 400 MHz for the devices listed above and 13 C-NMR spectra for the 75 MHz, 20 MHz or 100 MHz devices listed above.

The NMR values are delta values relative to TMS (tetramethylsilane) or D 2 O as a solvent relative to DSS (2,2-dimethyl-2-silapentane-5-sulfonic acid sodium salt).

Infrared (IR) spectra were recorded on a 15 Nicolet 5SX FT / IR instrument.

As far as chromatographic procedures are concerned, most solvents were Fischer HPLC grade. Ammonium acetate was purchased from Aldrich. The water was purified using a Barnstead NANOpure ™ water filtration system. The preparative chromatography of the organic compounds was carried out either by conventional gravity chromatography using conventional techniques or by flash chromatography as described by C. W. Still et al., J. Org. Chem. 43, 2923-24 (1978).

: '' 25 The following solvent mixtures were used:

Solvent mixture Ingredients 1 CHCl3SCH3OH: conc. NH 3 OH OH m.p. ratio 30 2: 2: i

A: A: A

• · 2 CHCl3: CH-aOK: conc. ΝΗ], 0Η vol. ratio

12: 11: 1 J

A: A: A

37 104898 H CHCl-a sCH ^ OH: conc. NHhOH ti 1: 4: 2: 10 ratio

A: V * V

CHClc: CH-aOH: Conc. NHnOH vol. ratio 5 3: 2: 10

A: A: A

6 CHCl3 CHOOH: conc. 7: 3: 1 NH 3 OH / v / v

A: A: A

γ Saline (0.85% 10 NaCl in distilled water): Conc.

v / v 4: 1 V: V

8 CHCl3: CHoOH: conc. NKhOK Ratio 4: 4: 1 0

A: A: A

15

The reported Rf values are obtained using these solvent mixtures and commercially available phase-silica gel TLC plates (GHLF 250 mieron, Analtech Inc. or Kieselgel 60F254, Merck). Preparative column chromatography was performed using Merck grade 60 silica gel (600 nm silica gel).

All percentages are by weight unless otherwise stated.

Some solids were dried using a rotary evaporator (Buchi 461) or a vacuum oven at about 55-60 ° C for a few hours. Additionally, an automatic Virti freeze-dryer (model 10-010) or a Speed Vac ™ concentrator was used to remove solvents.

Samarium-153 and lutetium-177 were produced in a research reactor at 30 University of Missouri (Columbia, MO). Yttrium-90 * ·; purchased from Oak Ridge National Laboratories.

1- (4-Isothiocyanate-benzyl) -diethylenetriaminipenta-acetic acid (SCN-Bz-DTPA) was prepared by a modification of the method of M. W. Brechbiel 35 et al., Inorg. Chem. 25, 2772-2781 (1986), and 381040889 were purified to obtain a single species by anion exchange HPLC (Q-Sepharose ™). Some of the chemicals used were obtained from the following sources: N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), free acid 5, and sodium salt, purchased from Behring Diagnostics (La.

Jolla, CA); sodium citrate (guaranteed) was from Fisher Scientific; thiophosgene was from Aldrich Chemicals; ammonium acetate, sodium acetate, ethylenediaminetetraacetic acid (EDTA), and phosphate buffered saline (PBS) were from Sigma

Diagnostics.

The HPLC columns used were: Hand-packed Q-Sepharose ™ (Pharmacia), either 1.5 cm x 25 cm or 2.5 cm x 25 cm; Zorbax ™ BIO Series 15 GF-250 (DuPont Instruments; 9.4mm x 25cm); Vydac ™ Protein C-4 (trademark of Separations Group, Hesperia, CA; 4.6mm x 25cm); and Mono-Q ™ and Sp-Sephadex ™ (trademark of Pharmacia Biotechnology Products). Sep-Pak ™ (trademark of Waters Associates) C-18 cartridges were purchased from 20 Waters Associates (Milford, MA), Sephadex ™ G-25 ker recycle columns (2.2 mL) from Isolab Inc. (Akron, OH) and Centricon ™ -30 (trademark of Amicon Division, WR Grace & Co., Danvers, MA) from Amico-n.

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

System I consisted of an LKB 2150 pump and 4152 control unit, a UV detector (LKB 2238 UV Cord), a Berthold LB 506 A HPLC radioactivity monitor 30 (International Berthold Group) and a Gilson 201-202 fraction *; from a catcher (Gilson International, Middleton, WI).

System II was equipped with an automatic sampler (WISP 710 B), two pumps (model 510) and an automatic gradient control (Waters Asso-35 ciates), a Perkin-Elmer LC-75 spectrophotometer detector, 39 104898

Beckman Model 170 radioisotope detector and fraction collector (Pharmacian Frac-100).

System III consisted of two Waters M-6000A pumps and a Waters 660 solvent programming device, a Waters 5 Model 481 variable wavelength detector, and a Waters Data Module; a ISCO fraction collector was also used preparatively.

System IV was a Dionex BioLC ™ system equipped with a UV detector with a wavelength range of 10 delta.

System V was a Waters Model 590 pump combined with an ISCO Model 2360 gradient programming device and a Dionex detector with variable wavelength.

A Sorvali 15 RT 6000B (DuPont cooled centrifuge) was used for centrifugation and concentration. A Speed Vac evaporator (Savant Instruments Inc., Hicksville, N.Y.) was used to remove volatile solvents. Radioactivity measurements were made on a Capintec ™ Radioisotope Calibrator (trademark of Capintec Inc.) on a CRC-12 instrument, a Beckman LS 9800 liquid scintillation counter (Beckman Instruments, Inc., Irvine, CA) or a Canberra 35 * multi-channel analyzer, with a 7.6 cm sodium iodide colocide floating in thallium.

In the examples of complex formation; The percent complex formation was determined by the following general exchange method, where indicated.

The 10 mL Plastic column was filled with 1-2 mL of SP-Sephadex ™ C-25 resin, which was swollen in water. Excess water was removed to the top of the column at the target point; come under pressure. The test solution (15 μΐ) was added to the resin, followed by the addition of 2 ml of isotonic saline and conc. NH4OH in a 4: 1 by volume ratio. The eluent was collected in counting tubes. Subsequently, 2 ml of additional eluent were used. After collecting the eluent 40 104898 in a second counting tube, compressed air was blown to the top of the resin to dry it. The dried resin was transferred to a third counting tube. Activity in each counting tube was measured with a Nal colloidal crystal counter connected to a Canberra Multichannel-5 Analyzer. The amount of metal present as a complex is expressed as the percentage (%) of total activity found in the eluent. The amount of metal in the free, uncomplexed state is expressed as the percentage of the total amount of metal that remains in the resin.

Removal of uncomplexed metal by ion exchange A 10 mL plastic column was filled with 0.5 mL of SP-Sephadex ™ C-25 resin swollen in water. Excess water was removed by centrifugation of the column for 15 min at 3000 rpm. 200 to 500 μΐ of complex solution was applied to the resin and the tube was centrifuged again for 3 minutes at 3000 rpm. The uncomplexed metal remained on the column and the complexed eluted with the solvent.

20 Preparation of the Yttrium Complex

The complexes were prepared by preparing 3 * 10 * 4 M aqueous solution of yttrium [YCl 3 * 6H 2 O, 303.26 g / mol, or Y (OAc) 3, 12.1% H 2 O]. Radioactive Y3 + solution (Oak Ridge: 25 National Laboratories) was added to achieve the desired level of radioactivity. To 990 µL of Y3 + solution was added • '10 μ 10 ligand solution (0.03 M) to give a 1: 1 molar ratio of ligand to metal. A 10-fold amount of ligand solution was used to form a 10: 1 ligand metal ratio. The pH was adjusted to 7.4 microliters with 30 volumes of HCl or NaOH. The solution was then examined * ·; the amount of complexed yttrium using the cationic exchange method described above.

Manufacture of a samarium complex

Samarium complexes were formed as described above for the yttrium-35 complexes except that 3 x 10 ~ 4 M

104898 samarium solution was prepared by dissolving Sm 2 O 3 (348.7 g / mol) in 0.1 M HCl. Radioactive Sm-153 was obtained from a research reactor at the University of Missouri (Columbia, Missouri) in approximately 3 x 10 -4 M solution in 0.1 M HCl.

5 Some terms used throughout this text are defined as follows.

Vocabulary: Pop. means strong; * OAc means an acetate group, 'OCOCH3; TLC refers to thin layer chromatography; DI H20 denotes deionized NANOpure ™ water; NANOpure ™ Water is pure water obtained from the Barnstead NANOpure ™ Water Filtration System; ambient temperature means a room temperature of 15, i.e., about 20-25 ° C; overnight means about 9-18 hours; LC stands for liquid chromatography; NBS means N-bromosuccinimide; MES stands for 2- (N-morpholino) ethanesulfonic acid; 20 HPLC refers to high performance liquid chromatography; PBS means Sigma-derived phosphate-buffered saline containing 120 mmol NaCl / L, 2.7 mmol KCl / L, and 10 mmol phosphate; 25 buffers (pH 7.4) / L.

SP-Sephadex ™ C-25 Resin is a cation exchange resin containing sulfonic acid functional groups and sold by Pharmacia, Inc .; min'1 = revolutions per minute; 30 pD represents the pH when hydrogen is replaced by deuterium; DOTA = 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid; HEPES = N-2-hydroxyethylpiperazine-N'-2-ethane-35 sulfonic acid; 42 104898 BFC = bifunctional chelator;

Cyclene = 1,4,7,10-tetraazacyclododecane; PA-DOTA = α- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; PA-DOTMA = α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1-acetic acid-4,7,10-tris (methyl acetic acid); BA-DOTA = α- (4-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid; 10 OMe-BA-DOTA = α - (5-amino-2-methoxyphenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; EA-D03A = 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid; DTPA = diethylenetriaminepentaacetic acid; SCN-Bz-DTPA = 1- (4-Isothiocyanate-benzyl) -diethylenetriaminepentaacetic acid; EDTA = ethylenediaminetetraacetic acid; the chelating agent is equivalent to ligand 20; the complex is equivalent to a chelate, the conjugate being a chelating agent or complex covalently bound to an antibody or antibody fragment; : 25 antibodies refer to CC-49, CC-83 and B72.3 and fragments thereof such as Fab and F (ab ') 2. Other possible antibodies are shown above. The hybridoma cell line B72.3 is deposited in the American Type Culture Collection under the accession number ATCC HB 8108, 30 and other designated murine monoclonal antibodies bind to epitopes of TAG-72, a tumor associated antigen.

The invention is illustrated in more detail by the following examples.

35 43 104898 Manufacture of starting materials

Example A

Preparation of DL-2-Bromo-4- (4-nitrophenyl) butanoic acid methyl ester To a solution of 5 ml of carbon tetrachloride and 15 ml (0.2 mol) of thionyl chloride was added 10.46 g (0.05 mol) of 4 (4-nitrophenyl) butanoic acid under a nitrogen atmosphere. The solution was refluxed for 1 hour, initially releasing hydrogen chloride gas and sulfur dioxide gas. To the warm solution was added 11.0 g (0.06 mol) of N-bromosuccinimide in 25 ml of carbon tetrachloride and three drops of 48% aqueous hydrogen bromide as catalyst. Bromine gas was found to be released. The dark red solution was refluxed for 35 minutes. The solution was cooled and poured into 100 ml of methanol with stirring. TLC analysis (ethyl acetate-hexane 60:40 by volume) showed the formation of a new product (R f = 0.69, silica gel plates). Excess solvent was removed by rotary evaporation and the dark red oil was filtered through a plug of flash chromatography-20 graphite silica gel (2.5 cm x 12.7 cm) using methylene chloride as eluent. Evaporation of the solvent gave a clear oil (yield: 15.43 g) which was a 85:15 mixture of the title compound and methyl ester of the butanoic acid starting material.

.. The title product was characterized by the following data: 1 H-NMR (CDCl 3): 8.16 (d), 7.38 (d), 4.20 (dd), 3.79 (s), 2, 88 (m), 2.38 (m): 13 C NMR (CDCl 3, 75 MHz): 169.6, 147.5, 129.3, 123.7, 53.0, 30 44.4, 35.5 , 33.0.

Example B

• · I

Preparation of 1-methyl ester of α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1-acetic acid

To a solution of 1.72 g (10.0 mmol) of 35 1,4,7,10-tetraazacyclododecane in 17 mL of pentene 44 104898-stabilized chloroform and stirred was added 2.07 g (5, 82 mmol) of DL-2-bromo-4- (4-nitrophenyl) butanoic acid methyl ester under nitrogen atmosphere with stirring. The reaction mixture was stirred at about 25 ° C for 48 hours. TLC (Analtech on silica gel plates using solvent mixture 2) showed the formation of the title product (Rf = 0.73). The yellow chloroform solution was applied to a 2.5 cm x 40 cm flash column of flash chromatography (silica gel eluted with 5% methanol containing si-10) eluted with 250 ml of 5% methanol in chloroform followed by solvent mixture 2. The fractions containing pure title product were combined and evaporated to give 2.15 g (5.46 mmol, 94%) of the title product as a pale yellow glass. Trituration of the chloroform solution of this vitreous material with diethyl ether resulted in the precipitation of an analytical white powder (m.p. 156-159 ° C) characterized by the following data: 1 H-NMR (CDCl 3): 8.14 (d), 7.39 ( d), 3.71 (s), 3.39 (dd), 2.5-3.0 (m), 2.08 (m), 2.01 (m); 13 C NMR (CDCl 3 75 MHz): 172.7, 149.3, 146.4, 129.2, 123.6, 62.3, 51.2, 48.9, 47.2, 45.8, 45 , 4, 32,8,30,9.

Example C

Preparation of 2-Bromo-2- (4-nitrophenyl) -acetic acid methyl ester

A mixture of p-nitrophenylacetic acid (25.0 g, 0.14 mol) and thionyl chloride (15.1 ml, 0.21 mol) in dry benzene (100 ml) was stirred and refluxed under N 2 for 3 hours. . The mixture was then evaporated to dryness under reduced pressure, the residue was dissolved in carbon tetrachloride, and the solution was stirred and refluxed under a nitrogen blanket. To the refluxing mixture was added bromine (7.2 mL, 0.14 mol) in small portions over 3 flow-35 rocks. The reaction mixture was allowed to cool, and methanol (50 mL) was slowly added to the oxygen chloride. The resulting bromo ester (27 g, 71%) was recovered as a yellow under reduced pressure through a 6 inch glass helical column by distillation (bp = 168 ° C at 1.1 mm pressure).

The structure of the title product was confirmed by the following spectral data: 1 H NMR (CDCl 3): 8.25 (d), 7.81 (d), 5.51 (s), 3.86 (s).

Example D

Preparation of 1-methyl ester of α- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-10-yl-1-acetic acid in 529 mg (2.068 mmol) of 2-bromo-2- (4-nitrophenyl) - acetic acid methyl ester (prepared according to the method of Example C) dissolved in 20 ml of acetonitrile was added in one portion a solution of 354 mg of 15 (2.068 mmol) of 1,4,7,10-tetraazacyclododecane in 20 ml of acetonitrile containing just enough methanol to achieve complete dissolution. The resulting pink solution was stirred for 1.5 hours, after which it was concentrated under reduced pressure at 35 ° C to a small volume. This crude monoester tetraamine was purified by silica gel chromatography using 20% (m / v) CH3OH in NH4OAc as the eluent. The purified monoester tetraamine isolated as the triacetate salt was then converted to the free amine.

· 270 mg of a salt dissolved in 3 ml of water were added at 0 ° C to an aqueous solution of K2CO3 (10% w / w) to bring the pH of the solution to 11. The basic solution was then extracted with 10 ml of chloroform ( five times), and the chloroform layers were combined, dried over sodium sulfate, and evaporated to give 160 mg (42% yield) of the desired monoester tetra-araine, which was characterized by NMR and FAB mass spectrometry ([M + H]). + = 366) and based on the following data: 46 10808 δ NMR (CDCl3): 8.12 (d), 7.44 (d), 4.75 (s), 3.76 (s) ), 2.67-2.44 (m); 13 C NMR (CDCl 3): 171.5, 147.6, 144.0, 130.0, 124.0, 67.0, 49.8, 47.9, 46.9, 46.0.

5 Example E

Preparation of N- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane

To a solution of chloroform (20 mL) containing 2.9 g (16.8 mmol) of 1,4,7,10-tetraazacyclododecane and stirred was added a solution of chloroform (20 mL) containing 2.1 g ( 8.5 mmol) of 2-methoxy-5-nitro-benzyl bromide. After stirring at room temperature for 3 hours, the reaction mixture was filtered and the filtrate was concentrated (under reduced pressure) to give an ice residue which was chromatographed on silica gel, solvent mixture 3). The monoalkylated product was isolated in 79% yield (mp = 154-156 ° C) and characterized by the following data: 13 C NMR (CDCl 3): 162.47, 140.63, 127.92, 125.49, 124.53 , 20 109.91, 55.88, 53.25, 50.90, 47.17, 45.45, 45.29.

Example F

Preparation of N- (2-hydroxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraazacyclododecane (2.3 g, 14 mmol). To a dioxane solution (20 ml) was added in one portion and under stirring a solution of 2-hydroxy-5-nitrobenzyl bromide (1.2 g, 7 mmol) in dioxane (15 ml). After a few minutes, K 2 CO 3 (1 g) was added and stirring was continued for 1 hour at room temperature. The reaction mixture was then filtered and the filtrate evaporated under reduced pressure to give a yellow semi-solid which was chromatographed (column) on silica gel using solvent mixture 5 as eluent. The desired monoalkylated product was isolated from the last third of a bright yellow eluent also. After evaporation, 104898, the yellow oil was triturated with CHCl3 (100 mL) and the mixture was filtered to remove the CHCl3-soluble amine starting material. The final product was then isolated in pure form as a yellow solid (1.2 g, 55%; mp = 142-515 ° C) and further characterized by 13 C NMR (D 2 O): 25.04. 25.28, 26.83, 31.80, 36.54, 101.88, 107.22, 110.92, 111.80, 115.26, 159.99.

Example 6 Preparation of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane

To a solution of 3.50 g (20.3 mmol) of 1,4,7,10-tetraazacyclododecane in 50 ml of pentene-stabilized chloroform was stirred, 4.00 g (17.4 g) was added dropwise over 5 minutes. mmol) 1- (2-bromoethyl) -4-nitrobenzene under a nitrogen atmosphere while stirring vigorously.

Stirring was continued for about 18 hours at room temperature (about 25 ° C), whereupon amine-20 hydrobromide crystals precipitated out of solution. The entire reaction mixture was loaded onto a flash chromatography column (2.5 cm x 46 cm) eluted with 5% methanol in chloroform; 200 ml of this solution was used as eluent and then elution was continued with solvent mixture 3. 1.45 g (9.7 mmol) of p-nitrostyrene (Rf = 0.98, solvent mixture 1) were separated from the desired product. The desired monofunctional title compound was isolated as a yellow oil (2.27 g, 7.06 mmol, 40.6%) which solidified on standing (Rf = 0.73, solvent mixture 1).

A sample of the title compound was recrystallized from CHCl3-cyclohexane and had the following characteristic characteristics: mp = 146.5-148.5 ° C; 1 H-NMR (CDCl 3): 8.135 (d, m), 7.395 (d, m), 2.91 (t), 2.77 35 (t), 2.72 (t), 2.50 (t) , 2.60 (s); 48 1 0 4 8 9 8 13 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 H

Preparation of 1- (4-nitrobenzyl) -1,4,7,10-tetraazacyclododode-5 cane To 50 ml of chloroform was added 3.5 g (20.3 mmol) of 1,4,7,10-tetra- azacyclododecane and 1.7 g (7.87 mmol) of p-nitrobenzyl bromide and the mixture was stirred under nitrogen at 25 ° C for 24 hours. The chloroform slurry of the hydrobromide salt 10 was then loaded onto a flash silica gel chromatography column (solvent mixture 3) of 2.5 cm x 43 cm size. 2.13 g (6.93 mmol) of the title product are obtained in the form of an analytical, light yellow solid in 88% yield (Rf = 0.58, solvent mixture 3), m.p. 15 128-129 ° C. - based on 1 H NMR (CDCl 3): 8.19 (d), 7.49 (d), 3.69 (s), 2.82 (t), 2.70 (t), 2.59 (m); 13 C NMR (CDCl 3, 75 MHz): 147.2, 128.4, 123.8, 58.8, 51.7, 20, 47.1, 46.3, 45.1.

(Example I is not at all to avoid confusion with subsequent biological example numbering.)

Example J

Preparation of 1- (4-aminobenzyl) 1,4,7,10-tetraazacyclododecane In 10 ml of methanol was dissolved 690 mg (2.25 mmol) of 1- (4-nitrobenzyl) -1,4, 7,10-Tetrazazacyclododecane (prepared by the method of Example H). 350 mg of catalyst containing 10% palladium on carbon were added to solution 30. Excess hydrogen was bubbled through the solution at 25 ° C. After 45 minutes, TLC showed the title product to be formed (Rf = 0.33, solvent mixture 3). The resulting title product 35 was purified by flash column chromatography on silica gel 49 104898 (solvent mixture 3), 2.5 cm x 41 cm, to give 480 mg (77%) of the title product as a pale yellow clear oil.

The title product in the form of the free base (103 mg, 0.37 mmol) was converted to the hydrochloride salt by blowing anhydrous hydrogen chloride through ethanol solutions of the free base. The yield of the resulting title compound, tetrahydrochloride salt, after washing with cold ethanol and vacuum drying, was 147 mg (0.347 mmol), m.p. 255-1060 ° C (dec.), And was further characterized by the following data: 1 H-NMR (D pD = 1.9): 7.56 (d), 7.47 (d), 3.95 (s), 3.28 (t), 3.23 (t), 3.09 (t), 2 , 96 (t); 13 C NMR (D 2 O, pD = 1.9, 75 MHz): 138.3, 134.2, 132.2, 126.0, 58.5, 50.3, 46.7, 44.6, 44 , 3.

Example K

Preparation of 2-methoxy-5-nitrobenzyl nitrile To a solution of sodium cyanide (6 g, 121.9 mmol) in water (5.3 mL) at 70 ° C was added in small portions while stirring, a hot solution containing 2 methoxy-5-nitrobenzyl bromide (25 g, 101.6 mmol) in ethanol (15 mL). The reaction mixture was then refluxed for 1.5 hours and cooled and filtered. The filter cake was washed with acetonitrile and the filtrate was evaporated under reduced pressure to give 2-methoxy-5-nitrobenzyl nitrile (19.5 g, 100%) as a tan solid. The product was characterized by the following data: 13 C NMR (CDCl 3): 161.4, 141.0, 125.7, 124.6, 119.8, 116.5, 30 110.2, 56.3, 18.8.

Example L

Preparation of 2-methoxy-5-nitrophenylacetic acid A slurry containing 2-methoxy-5-nitrobenzyl nitrile (19.5 g) (prepared according to the procedure of Example K) was concentrated. HCl (20 mL) was refluxed for 3 104898 hours. After cooling, the crude product was recovered by filtration and washed with water. The solid was mixed with hot aqueous sodium hydroxide and the mixture filtered while hot. The orange solution was cooled and acidified with HCl. The precipitate was filtered off, washed with water and dried to give 2-methoxy-5-nitro-phenylacetic acid as a white solid (15 g, 70%). The product was characterized by the following data: ¹³C NMR (CDCl3-CD3OD): 172.8, 162.5, 140.7, 126.2, 124.7, 124.1, 109.7, 55.8, 35, 0.

Example H

Preparation of α-Bromo (2-methoxy-5-nitrophenyl) acetic acid methyl ester In a slurry of 2-methoxy-5-nitrophenylacetic acid (2.266 g, 10.74 mmol) (prepared according to the procedure of Example L) in dry benzene (50 mL), thionyl chloride (4.0 mL, 54.8 mmol) was added. A drying tube and condenser were placed in the flask and the mixture was refluxed for 2 hours. The resulting yellow solution was concentrated under reduced pressure to a small volume and mixed with dry carbon tetrachloride. Bromine (0.6 mL, 11.6 mmol) was added and the solution refluxed for 2 days, protected from atmospheric moisture. The reaction mixture was concentrated to a small volume and methanol (50 mL) was added. After evaporation of excess methanol under reduced pressure, the resulting oil was purified by chromatography (silica gel, 4: 1 methylene chloride-hexane). The title product (2.399 g, 30 74%) was obtained as a yellow oil. The product was characterized by the following data: 1 H NMR (CDCl 3): 8.45 (dd), 8.15 (dd), 6.95 (d), 5.75 (s), 3.99 (s), 3, 78 (s).

104898

Example N

Preparation of α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1-acetic acid methyl ester

To a solution of 1,4,7,10-tetraazacyclo-5-decane (2.72 g, 15.79 mmol) in chloroform (50 mL), which was stirred, was added a solution of o-bromo (2- methoxy-5-nitrophenyl) acetic acid (2.399 g, 7.89 mmol) (prepared by the procedure of Example M) in chloroform (10 mL). The mixture was stirred at room temperature for 2 hours. The cloudy mixture was then concentrated to a small volume under reduced pressure and at room temperature. = the crude product was purified by chromatography (silica gel, solvent mixture 3). The title compound was recovered as a yellow solid (2.60 g, 83%) and characterized by 13 C NMR (CDCl 3): 170.7, 161.6, 139.9, 125.0, 124 , 5, 124.2, 110.2, 59.5, 55.5, 50.5, 48.0, 47.3, 45.6, 44.3, 43.5.

Example 0 Preparation of isopropyl 20-ester of DL-2-bromo-4- (4-nitrophenyl) butanoic acid

To a solution of carbon tetrachloride (10 mL) and thionyl chloride (30 mL, 0.4 mol) was added 4- (4-nitrophenyl) butanoic acid (21.0 g, 0.10 mol) under H 2 O, under a nitrogen atmosphere. [J. Org. Chem. 40, 3420-7 (1975); 25 27]. The solution was refluxed for 1 hour at which time hydrogen chloride and sulfur dioxide were rapidly released. At this point, N-bromosuccinimide (22.0 g, 0.12 mol) dissolved in carbon tetrachloride (50 mL) was added to the warm solution and 8 drops of a 48% aqueous hydrogen bromide solution were observed to release bromine. The dark red solution was refluxed for an additional 35 minutes. The solution was cooled and poured into iso-propanol (400 mL) with stirring. TLC analysis (methylene chloride, silica gel plates) revealed a new product 35 (Rf = 0.73). Excess solvent was removed and the dark brown oil was filtered through a pad of flash chromatography silica gel (7.6 cm x 15 cm) using methylene chloride as eluent. The solvent was removed under reduced pressure and the light yellow oil was transferred to a flash chromatography column 5 (7.6 cm x 46 cm) and eluted with methylene chloride to give 25.0 g (0.076 mol) of the title bromoester product in 75% yield as a clear oil in isopropanol. solvated with a product containing less than 5% non-brominated ester and characterized by: 1 H-NMR (CDCl 3): 8.16 (d, 2H), 7.38 (d, 2H), 5.05 (Sept, 1H), 4.14 (dd, 1H), 2.88 (m, 4H), 2.39 (m, 4H), 1.29 (d, 6H) ; 13 C NMR (CDCl 3): 168.7, 147.7, 129.3, 123.8, 69.9, 45.1, 15 35.6, 33.0, 21.5, 21.2.

Example P

Preparation of 1 - methyl ester of α - [3- (4-nitrophenyl) propyl] -1,4,7,10-tetraaza-cyclododecane-1-acetic acid

To a solution of 3.137 g (18.2 mmol) of 20 cyclenic free base in 30 ml of pentene-stabilized chloroform was stirred, 4.81 g (14.6 mmol) of DL-2-bromo-4- (4 -nitrophenyl) butanoic acid isopropyl ester under a nitrogen atmosphere while stirring. The reaction solution was stirred for 24 hours; · 25 at room temperature. TLC analysis (solvent mixture 2) revealed conversion to the title monoalkylation product (Rf = 0.78, detection by ninhydrin, iodine and UV light). The yellow chloroform solution was transferred to a 2.5 cm x 43 cm flash column of silica gel 30 pre-eluted with 5% methanol in chloroform. Elution with 300 ml of this solvent mixture followed by solvent mixture 2 gave fractions containing pure title compound which were combined and evaporated to give 4.85 g of 35 (5.46 mmol) of the title compound as a free base. of the product as a light oil (79% yield), which was further characterized by 1 H NMR (CDCl 3): 8.15 (d, 2H), 7.40 (d, 2H), 5.07 (p, 1H), 3.35 (dd, 1H), 2.65-3.0 (m, 13H), 2.5-2.64 (m, 5H), 2.14 (m, 1 H), 2.00 (m, 1H), 1.28 (dd, 6H); 13 C NMR (CDCl 3): 171.6, 149.5, 146.5, 129.2, 123.6, 68.1, 62.7, 49.2, 47.5, 45.9, 45.7 , 32.9, 31.0, 22.1, 22.0; IR (CDCl 3, cm -1): 3231 (N-H), 2978, 2933, 1721 (ester carbonyl), 1601, 1512, 1458, 1345, 1107; FAB mass spectrum (m / e): 422 ([M + H] +), 408, 392.

Preparation of Compounds of Formula IA

Example 1a Preparation of monomethyl triethyl ester of α- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid in 46 ml of acetonitrile containing 4.6 g of , 3 mmol) of freshly powdered anhydrous potassium carbonate, 700 mg (1.91 mmol) of 1-methyl-20-ester of α- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1-acetic acid (prepared according to (D). To this suspension was added at one time 1.022 g (6.12 mmol) of ethyl bromoacetate. After stirring at room temperature for 4 hours, the suspension was filtered under reduced pressure, the filter cake was washed with 15 ml .. 25 acetonitrile (twice) and the filtrate was evaporated under reduced pressure at 38 ° C to give the crude title tetraester as a purple solid. The tetraester was then purified by silica gel chromatography using 5% C2H5OH in CHCl3 followed by solvent mixture 3 to give 620 mg (0.988 mmol, 52%) of the desired product. This product was characterized by FAB mass spectrometry ([M + H] + = 624) and further by 1 H-NMR (CDCl 3): 8.24 (d), 7.45 (d), 4.94 ( s), 4.35 - 4.10 35 (m), 3.79 (s), 3.75 - 1.84 (m), 1.34 - 1.22 (m); 54 104898 13C NMR (CDCl3): 174.3, 173.8, 173.6, 171.5, 147.6, 138.9, 131.5, 123.4, 64.8, 61.5, 61 , 4, 60.2, 55.4, 55.0, 53.1, 52.9, 52.7, 52.4, 51.9, 51.7, 48.8, 48.3, 44.9 , 19.1, 14.1.

Example 2 Preparation of o- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid In 30 ml of 6 N HCl was dissolved 300 mg ( 0.478 mmol) of m - (4-nitrophenyl-1,4,7,10-tetraazacyclododecane-10,4,7,10-tetraacetic acid monomethyl triethyl ester (prepared according to the procedure of Example 1) and refluxed overnight. the solution was evaporated under reduced pressure at 70 ° C to give a yellow solid This solid 15 was dissolved in 3 mL of water, filtered and the filtrate evaporated to give 253 mg (0.378 mmol, 79%) of crude product. [silica gel, development with 20% (w / v) CH3OH in NH4OAc] and characterized by FAB mass spectra-20 metric ([M + H] + = 526) and further by the following data: Ή-NMR ( D20): 8.21 (d), 7.42 (d), 483 (s), 3.83-2.19 (m), 1.92 (s); 13 C NMR (D 2 O): 175.0 , 173.6, 171.8, 1 67.0, 166.3, 146.9, 25 138.3, 130.1, 123.0, 62.2, 54.0, 53.0, 52.2, 50.4, 97.3, 46 , 8, 44.8, 42.8, 20.0.

Example 3 Preparation of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (BA-DOTA) Method A:

Catalytic hydrogenation of the nitro group of the tetra acid (prepared by the procedure of Example 2) was carried out using an Adams catalyst (PtO 2) and hydrogen in approximately quantitative yield to afford the title product. This product was characterized by 55,104,898 FAB mass spectrometry ([M + H] + = 496) and anion exchange HPLC (Q-Sepharose ™ gel), and additionally by the following data:? -NMR (D 2 O):? 12 (d), 7.86 (d), 5.66 (s), 4.73 - 4.57 δ (m), 4.27 - 3.78 (m), 3.39 - 2.91 ( m); 13 C NMR (D 2 O): 176.54, 176.52, 176.46, 141.1, 128.9, 120.9, 112.5, 65.0, 55.9, 55.7, 53.6 , 49.7, 49.5, 49.3, 45.7, 45.4, 44.9, 44.6, 41.4.

Method B: Another process for the preparation of this compound was the conversion of the monoester tetraamine compound obtained in Example D to the mono acid tetraamine compound (6N HCl, reflux overnight) followed by aqueous alkylation with bromoacetic acid and then reduction of the nitro group to the amine group identical to the product described above.

Example 4 Preparation of α- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid In 60 ml of 6 N HCl was dissolved 2.0 g (5, 48 mmol) of the monoester tetraamine compound (prepared according to the procedure of Example D). The mixture was heated at 95 ° C overnight (about 10 hours) then evaporated under reduced pressure at 75 ° C to give 2.53 g of 25 (5.11 mmol, 96%) of the mono acid tetraamine tetrahydrochloride as a yellow solid.

The above mono acid tetraamine tetrahydrochloride (0.5 g, 1.0 mmol) was dissolved in 15 mL of water and the pH was adjusted to 9 with NaOH. A separate solution of bromo-30 acetic acid (0.71 g, 5.13 mmol) was prepared. , and was added to an aqueous solution of mono-acid tetraamine. The pH of the solution was again adjusted to 9 and maintained at this value during the reaction with small additions of 1.0 N NaOH. While the reaction was stirred, aliquots were sampled and the progress of the alkylation monitored by anion exchange HPLC 104898. When the amount of dialkylation product (3 acetate groups present in total) had reached its maximum, the entire reaction mixture was lyophilized to give a dark solid. This crude mixture of alkylation products was then purified by silica gel chromatography (elution with solvent mixture) followed by preparative anion exchange chromatography (elution from 0% to 100% in 1M NH4OAc) followed by preparative development on TLC plate. solvent mixture 4) and finally 10 silica gel chromatography (elution with solvent mixture 4).

This extensive purification procedure afforded 30 mg (0.06 mmol, 6.0%) of the title product as a yellow solid. This product was characterized by FAB mass spectrometry ((M + H] + = 468) and anion exchange HPLC 15 and further by 1 H-NMR (D 2 O): 8.12 (bs), 7.35 (bs) , 4.76 (s), 3.57 - 3.44 (m), 2.97 - 1.83 (m), 1.80 (s); 13 C NMR (D 2 O): 181.75, 180, 68, 179.00, 175.37, 147.70, 141.22, 133.08, 123.53, 68.44, 59.72, 56.00, 52.80, 49.96, 20 49.29 , 47.86, 45.36, 44.26, 42.78, 42.25, 23.72.

Example 5 α- (4-Aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid pentahydrochloride and α- (4-aminophenyl) -1,4,7,10-tetra- Preparation of 25 Acacyclododecane-1,4,10-Tetraethyl · 25 Acid Cyclopentahydrochloride

The crude mixture of starting products was prepared according to the procedure described in Example 4. This crude mixture was chromatographed on a silica gel column using 20% (w / v) CH 3 OH in NH 4 OAc as eluent. The appropriate fractions from this column were combined and evaporated to dryness. The crude product containing significant amounts of NH4OAc was dissolved in 80 mL of NANOpure ™ water and freeze-dried to give a light brown solid. This solid was dissolved in 20 mL of NANOpure ™ -35 water, and the solution was shaken with PtO 2 in 104898 H2 atmosphere until hydrogen consumption ceased. The catalyst was separated by vacuum filtration and the filtrate was lyophilized to give a yellow solid. The solid was dissolved in 3 mL of solvent mixture 4 and hand-chromatographed on silica gel using solvent mixture 4. The combined fractions were then stripped of solvent under reduced pressure and freeze-dried to give 779.6 mg of a pale yellow solid. The 13 C NMR and proton NMR spectra indicated that this product is a 50:50 mixture of 10 possible geometric isomers. The isomer mixture was treated with HCl (excess) and lyophilized to give 548.4 mg (61% yield) of the title product, m.p. > 270 ° C. Anion exchange chromatography (HPLC system III) showed only one major peak (purity> 94%); The FAB mass spectrum revealed the presence of geometric isomers ([M + H] * = 438).

Example 6 1,4,7,10-Tetrame-20-ethyl ester of α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid manufacturing

A solution of 1.43 g (3.63 mol) of α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1-acetic acid 1-methyl ester (prepared according to Example B procedure) in 40 mL of argon purged 25 acetonitrile, 1.71 g (12.34 mmol) of anhydrous potassium carbonate was added with vigorous stirring. 1.78 g (11.61 mmol) of methyl bromoacetate were added to the reaction mixture, and the reaction mixture was stirred at about 25 ° C for 48 hours. TLC analysis showed a new product was formed (Rf = 0.62, solvent mixture 2). 6.0 g of flash chromatography silica gel were added to the slurry and the acetonitrile was removed on a rotary evaporator. The resulting material was applied to a 2.5 cm x 41 cm silica gel column pre-eluted with chloro-35 form containing 5% methanol. About 400 ml of the prepared solution was used to elute 58 104898 of a number of polar impurities and unreacted alkylating agent. Elution with solvent mixture 2 was then carried out and the fractions containing product (R f = 0.62) were collected, combined and evaporated to give 2.1 g (3.44 mmol, 95%) of the title product as a pale yellow glass. 1 H NMR showed this product to be a 2: 1 mixture of conformation isomers, i.e. geometric isomers.

1 H-NMR (CDCl 3, 50 ° C): 8.137 (d), 8.128 (d), 7.398 (d), 10 7.385 (d), 3.82 (s), 3.76 (s), 3.75 ( s), 3.74 (s), 3.68 (s), 1.5-3.52 (m); 13 C NMR (CDCl 3, 50 ° C, 75 MHz): 175.8, 174.2, 174.1, 174.0, 149.0, 129.5, 129.3, 123.6, 60.1, 55.1, 52.8, 52.7, 52.6, 52.4, 52.2, 52.1, 52.0, 51.2, 51.1, 51.0, 49.1, 48, 8, 15 47.5, 45.2, 34.2, 32.6.

Example 7 Preparation of o- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid 1,4,7,10-tetramethyl ester In 25 ml of methanol containing 400 mg of catalyst containing 10% palladium on carbon, 1.41 g (2.31 mol) of α- [2- (4-nitrophenyl) ethyl] -1 was dissolved under a nitrogen atmosphere. , 4,7,10-Tetrazazacyclododecane-1,4,7,10-tetraacetic acid 1,4,7,10-tetramethyl ester; 25 (prepared according to the procedure of Example 6).

Excess hydrogen was bubbled through the solution at 1 bar for 3 hours. TLC showed strongly formation of a ninhydrin-like substance (Rf = 0.62, solvent mixture 2).

The methanol-containing catalyst slurry was filtered through silica and the solvent evaporated to give the title product (1.21 g, 2.08 mmol, 91%) as a white solid (2: 1 mixture of conformation isomers). The minor conformation isomer, i.e. the geometric isomer, was separated from the main isomer by flash-35 chromatography using 10% methanol in chloroform containing 59 104898 to give the title product as an off-white powder (m.p. 89-95 ° C) which was characterized by: CDCl3 / 50 ° C): 6.94 (d), 6.89 (d), 6.69 (d), 6.66 (d), 3.91 (S), 3.80 (s), 3.78 (s), 3.74 (s), 3.73 (s), 3.72 (s), 3.71 (s), 1.5-3.5 (m); 13 C NMR (CDCl 3, 50 ° C, 75 MHz): 176.1, 174.0, 173.9, 172.2, 170.4, 169.6, 144.2, 144.1, 130.6, 130.5, 129.5, 129.1, 115.9, 115.8, 62.6, 58.7, 55.1, 54.3, 52.7, 52.5, 52.3, 10 52 , 2, 52.1, 52.0, 51.9, 51.8, 51.7, 50.2, 50.0, 47.3, 44.7, 32.8, 31.8, 30.0, 25 2; FAB mass spectrum (m / e): 602 ([M + Na +] +), 618 ([M + K +] +), 624 ([M + 2Na + - H +] +).

Example 8 Preparation of 15 o- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (PA-DOTA) in 50 ml conc. . Hydrochloric acid was dissolved in 1.5 g (2.59 mmol) of α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetra-20-azacyclododecane-1,4,7,10-tetraacetic acid 1,4 , 7,10-tetramethyl ester (2: 1 crude mixture prepared according to the procedure of Example 7). The reaction mixture was refluxed at about 105 ° C for 6 hours. TLC (solvent mixture 1) showed an alteration of the ester (Rf = 0.88) with the hydrochloride salt; · To the title product in the form of 25 lan (Rf = 0.43). Excess solvent was removed on a rotary evaporator and the resulting white solid was dried. 1.6 g of the title product was obtained as the hydrochloride salt and characterized by the following data: "1 H-NMR (D 2 O, pD = 1.0, 90 ° C): 7.48 (d), 7.42 (d), 2.8 - 4.3 (m), 2.23 (m), 2.03 (m); 13 C NMR (D 2 O, pD = 1.0, 90 ° C, 75 MHz): 176 , 4, 174.9, 171.6, 144.7, 132.9,, 132.8, 130.4, 125.7, 61.7, 56.8, 56.0, 35, 53.7, 53.1 , 51.6, 47.9, 34.3, 37.6;

6 O

104898 FAB mass spectrum (m / e): 524 ([M + H] +), 546 ([M + Na +] +, 624 ([M + 2Na + - H +] +).

Any minor ester impurities were removed by flash chromatography and solvent mixture 1 from the title compound in the form of the hydrochloride salt. 1.0 g of the hydrochloride salt of the title compound was transferred to a 2.5 cm x 58 cm flash silica gel chromatography column. The fractions containing only the title product in the form of the ammonium potassium mixed salt 10 were combined to give 580 mg of the product.

HPLC analysis showed that the surface area purity of the substance was greater than 98% at 230 and 254 nm.

FAB mass spectrum (m / e): 562 ([M + K *] *), 584 ([M + K + + Na * - H +] +), 624 ([M + 2Na + - H +] +).

Example 9 Isolation of ammonium potassium mixed salt of o- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,10-triacetic acid

A crude 20 solution of mixed tetra and tri-acids (2.05 g) (prepared according to the procedure of Example 8) was dissolved in the minimum amount of solvent mixture 1 and transferred to a 30 cm x 7.6 cm size column of flash silica gel. pre-eluted with this solvent. Eluted fractions containing the title product 25 (Rf = 0.63 in solvent mixture 1) were collected to give 200 mg of α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4, 10-Triacetic acid, ammonium potassium mixed salt. 1 H-NMR and 13 C-NMR analysis indicated that this triic acid was a symmetric isomer 30 (1,4,10-tri-acid position isomer): 1 H-NMR (D 2 O, pD = 0.5 with DCl) adjusted, T = 90 ° C): 7.52 (d), 7.46 (d), 3.60 (m), 3.54 (m), 3.19 (m); 13 C NMR (D 2 O, pD = 0.5, T = 90 ° C): 176.1, 170.2, 140.0, 132.7, 131.1, 126.1, 57.6, 57.2 , 56.1, 54.9, 53.2, 51.5, 35 51.2, 31.2.

610 104898 FAB mass spectrum (m / e): 466 ([M + H +] +), 488 ([M +

Na *] +), 504 ([M + K +] +), 526 ([M + K + + Na + - IT] +) f 542 ([M + 2K + - H +] +).

Example 10 Preparation of tetramethyl ester of 5α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

A mixture of α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1-acetic acid 1-Melo style ester (580 mg, 1.47 mmol) (prepared (Example N)) and powdered potassium carbonate (1.15 g, 8.32 mmol) in acetonitrile (20 mL) and stirred, methyl bromoacetate (565 μΐ, 5.97 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The mixture was then filtered and the mixture concentrated to an oil under reduced pressure. The crude product was purified by chromatography (silica gel, 8% methanol in methylene chloride). The title product was obtained as a yellow solid (469 mg, 52%; TLC R f = 0.4, silica-20 gel, 10% CH 3 OH in CHCl 3) and further characterized by 13 C NMR (CDCl 3): 173 , 2, 172.6, 161.2, 139.1, 125.1, 124.6, 120.5, 110.7, 57.0, 55.6, 53.5, 52.6, 51.3 , 50.8, 46.8, 46.0, 44.0.

: Example 11 Preparation of α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid

A solution of α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-30-methyl ester (prepared according to the procedure of Example 10) in anhydrous HCl (5 mL, JT Baker ULTREX) was refluxed under nitrogen for 5 hours. The solution was then evaporated to dryness under reduced pressure to leave a solid residue. This residue was purified by chromatography (silica gel, solvent mixture 6) to give the title product as an off-white solid (209 mg). This material was converted to the tetrahydrochloride salt and characterized by the following data: 13 C NMR (D 2 O-DClf 80 ° C): 171.0, 166.9, 161.2, 139.0, 125.5, 5 119.3, 110.7 , 56.8, 54.5, 52.7, 51.0, 49.2, 49.0, 46.3, 42.9.

Example 12 Preparation of the tetraammonium-10 salt of α- (2-methoxy-5-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid

Of a solution of α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (157 mg; prepared according to the procedure of Example 11). ) in water (20 mL), platinum oxide 15 (20 mg) was added. This mixture was hydrogenated (hydrogen pressure 1 bar) at room temperature for 1 hour. After filtration, the material was further purified by chromatography (silica gel, solvent mixture 5) to give the title compound (141 mg) as an off-white solid, which Karak-20 was triturated with 13 C NMR (D 10 -DCl): 175.5; 172.6, 172.3, 159.9, 128.8, 127.5, 124.6, 123.8, 115.5, 61.2, 58.1, 57.3, 55.7, 53.4, 51.6, 49.6, 47.4.

Example 13: Preparation of 1- (4-aminobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid 4,7,10-trimethyl ester in 4 ml of argon purged acetonitrile with stirring 100 mg (0.236 mmol) of the tetrahydrochloride salt of 1- (4-amino-30-benzyl) -1,4,7,10-tetraazacyclododecane (prepared according to the procedure of Example J), 260 mg (1.88 mmol) of potassium carbonate, and 108 mg (0.708 mmol) of methyl bromoacetate. The mixture was stirred at 25 ° C for 48 hours. The salt-containing solution was transferred to a flash silica gel chromatography column of 63-104898 size 2.5 cm x 10 cm and eluted with acetonitrile. The fractions containing the desired product were combined and the solvent removed by rotary evaporation to give 65 mg (0.132 mmol, 56%) of the title product, which was further characterized by Karak-5 based on 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 (s), 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, 10 115.0, 55.9, 55.3, 54.7, 53.1 , 51.7, 51.4, 50.6, 50.5, 47.4.

Example 14 Preparation of the hydrochloride salt of 1- (4-aminobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-trietic acetic acid A solution of 42 mg (0.085 mmol) of 1- (4-amino) benzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-tri-acetic acid 4,7,10-trimethyl ester (prepared according to the procedure of Example 13) in 2 ml of 6N hydrochloric acid, heated to 80 ° C. To C for 2 hours. TLC revealed a number of products (Rf = 0.60, solvent mixture 1, title product). Removal of the solvent gave 41 mg of the crude title product.

Example 15 Preparation of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-25-cyclododecane-4,7,10-triacetic acid 4,7,10-trimethyl ester

To a solution of 2.24 g (6.97 mmol) of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane (prepared by the procedure of Example G) in 75 ml of acetonitrile purged with argon, 3.17 g of anhydrous potassium carbonate were added with vigorous stirring. To this reaction mixture, 3.20 g (20.9 mmol) of methyl bromoacetate was added dropwise over 5 minutes. The reaction mixture was stirred for 17 hours under argon.

TLC analysis showed the starting material (Rf = 0.73, solvent mixture 1) 64 104898 to be converted to the new product (Rf = 0.46, solvent mixture 1). 70 ml of chloroform was added to the solution, and the solution containing the suspended salt was transferred to a 2.5 cm x 18 cm flash silica gel chromatography column. The product 5 was eluted with 10% methanol in chloroform to give 2.95 g of the title product as an amber oil which formed a brittle vitreous material under reduced pressure (78%). The title product was characterized by the following data: 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 16 Preparation of 4,7,10-methyl ester of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid in 50 ml of methanol 2.8 g (5.18 mmol) of 20 crude 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triethetic acid were dissolved in 4,7,10 -tri-methyl ester (prepared according to the procedure of Example 15). To the stirred and degassed solution of nitrogen was added catalyst (600 mg); '25 containing 10% palladium on carbon. The solution was kept under a nitrogen atmosphere and hydrogen (1 bar, 20 -25 ° C) was then passed through the solution with stirring for 2.5 hours. Nitrogen was then passed through the solution for several minutes, and the catalyst was removed by filtration through a thin pad of diatomaceous earth. TLC analysis ·· (10% methanol in chloroform) showed the presence of the title product (Rf = 0.14). The title product was eluted from silica gel with chloroform to give 2.2 g (4.33 mmol, 83%) of a yellow vitreous which was ka-35 structured based on the following information: 104898 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, 5 47.8, 28.6; FAB mass spectrum (m / e): 508 ([M + H] +).

Example 17 Preparation of the hydrochloride salt of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid (EA-D03A) in 30 ml conc. HCl was dissolved in 850 mg (1.69 mmol) of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid 4.7, 10-trimethyl ester (prepared according to the procedure of Example 16). Solution-15 was stirred at 70-80 ° C for 2.5 hours, allowed to cool to 25 ° C with stirring, and stirring was continued for about 18 hours. The solvent was removed by rotary evaporator under reduced pressure (10 mm, 75 ° C). The solvent and excess hydrogen chloride were removed from the resulting clear oil by vacuum drying (0.1 mm, 45 ° C). The title product was obtained as 820 mg as a white solid (Rf = 0.46, solvent mixture 2), which was further characterized by 1 H-NMR (D 2 O, pD = 1.9, T = 90 ° C): 7.50 (d), 7.41 (d), 4.26: 25 (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 mass spectrum (m / e): 466 ([M + H +] +), 488 ([M + Na *] +), 30510 (M + 2Na + - H +] +).

HPLC analysis showed area purity above 92% (254 nm) on a 10 cm Partisil-5 OD53 RAC II reverse phase column. The eluent was 0.05 M potassium phosphate solution, pH 7.0 containing 10% acetonitrile.

35 66 104898

Example 18 Preparation of 1- [2- (4-Isothiocyanate-Phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (SCN-EA-D03A) 5 1- [2- (4) -aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid hydrochloride salt (prepared according to the procedure of Example 17; 106 mg, 0.21 mmol) was dissolved in 2 ml of water under nitrogen gas and stirring. Sodium hydrogen carbonate (105.6 mg, 1.26 mmol) was slowly added to prevent foaming due to the evolution of carbon dioxide (resulting in a pH of 8.0). Thiophosgene (16.8 μΐ, 0.22 mmol) was added, and after 1 hour vigorous stirring, TLC analysis (20% water in acetonitrile) showed the starting material (Rf = 0.12) to be converted to the title product (Rf = 0, 26). The solvent was removed from the crude product by rotary evaporation to give 130 mg of the title product containing sodium chloride. Infrared analysis of this material (KBr tablet) confirmed the presence of the isothiocyanate group (SCN = 2150 cm -1).

Example 19 Preparation of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid triethyl ester; 25 ml of a solution of acetonitrile containing 395 mg (1.12 mmol) of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane (prepared according to the procedure of Example G) 1, 5 g (11 mmol) of K 2 CO 3 and stirred were added in one portion 496 μΐ (4.5 mmol) of ethyl 30 bromoacetate. After heating the mixture at 68 ° C under N 2 for 1 hour, the resulting suspension was filtered. The filter cake was washed with CH 3 CN (2 x 10 mL). The filtrate was then evaporated to give the crude product as a viscous oil which was triturated with diethyl 35 ether (30 mL) and evaporated to give 650 mg (100% yield) of 1- [2- (4-nitrophenyl) ethyl]. -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid triethyl ester which was characterized by the following data: 5 H-NMR (CDCl 3): 8.2 (d), 7.6 (d), 4, 3 (q), 2.6 - 3.8 (m), 1.55 (t); FAB Mass Spectrum: [M + H] + = 588.

Example 20 Preparation of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazalo-cyclododecane-4,7,10-triacetic acid

To a suspension of 200 mg (0.35 mmol) of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid triethyl ester (prepared according to Example 19). procedure) in 15 mL of water, 59 μΐ NaOH (50 wt%, 3 equiv) was added. The mixture was stirred at 80 ° C for 6 hours. The resulting homogeneous orange solution was then cooled to room temperature and freeze-dried to give an orange solid which was purified by HPLC (Q-Sepharose ™, HPLC System III) using a linear 60 minute solvent gradient of 0 to 10% HOAc: aqueous solution to give (50%) the hydrolyzed product 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclo-dodecane-4,7,10-trietic acetic acid, which was characterized by: based on the following data: 1 H-NMR (D 2 O): 8.22 (d), 7.55 (d), 3.60 - 3.90 (m), 3.10 - 3.40 (m).

Example 21 Preparation of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-30-cyclododecane-4,7,10-triacetic acid (EA-D03A)

50 ml of an aqueous solution of 214 mg (0.43 mmol) of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10- trietic acetic acid (prepared by the procedure 35 of Example 20) and 40 mg of 10% Pd / C catalyst. The suspension was shaken until hydrogen consumption ceased. After filtration, the aqueous solution was lyophilized to give 197 mg (98%) of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid as a tan solid. as a substance which was characterized by the following data: 1 H-NMR (D 2 O): 7.35 (d), 7.20 (d), 3.10-3.70 (m); 13 C NMR (D 2 O): 173.85, 172.50, 137.90, 131.90, 130.22, 121.55, 56.25, 55.14, 54.42, 50.12, 49.65 , 49.31, 31.00.

Example 22 Preparation of 1- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid 4,7,10-trimethyl ester

To a solution of acetonitrile (20 mL) containing 1.0 g of 15 (3 mmol) of 1- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane (prepared according to the procedure of Example E) was added together batch 1.6 g (11 mmol) of methyl bromoacetate. After 1 hour, potassium carbonate (0.3 g) was added and stirring was continued at room temperature for 12 hours. The reaction mixture was then filtered and the filtrate concentrated (under reduced pressure). The residue was subjected to column chromatography (silica gel, CH 3 CN-CH 3 OH 9: 1 by volume) to give the triester as a white solid after evaporation in a 68% yield and characterized by 1 H-NMR (CDCl 3): 8.19 (d), 8.14 (s), 7.18 (d), 2.58 - 3.99 (m); 1 H NMR (CDCl 3): 173.53, 172.83, 164.59, 142.28, 128.51, 30 127.69, 126.49, 112.30, 57.25, 56.90, 55, 03, 54.21, 53.06, 52.09, 52.00, 51.54, 50.68, 50.30, 49.85, 49.63, 49.31, 49.00, 48.68, 48.37, 48.05, 47.70, 47.39.

Example 23 Preparation of 1- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraaza-35-cyclododecane-4,7,10-triacetic acid 69 104898 in 6 M hydrochloric acid solution (3 ml) which containing 0.25 g (0.45 mmol) of 1- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid 4.7.10-trimethyl ester (prepared according to Example 22). procedure), stirred and heated at 100 ° C for 24 hours. After cooling to room temperature, 5 mL of water was added and the aqueous solution was lyophilized to give a tan solid. Tri-acid from Pyl HPLC (silica gel, solvent mixture 5) was isolated as an off-white solid (m.p. 228-230 ° C, dec.) In 60% yield and further characterized by the following data: 1 H-NMR (D 2 O): 8.18 (s) ), 8.09 (s), 7.12 (d), 3.87 (s), 2.10 - 3.60 (m); 13 C-NMR (D 2 O): 180.45, 179.80, 163.66, 140.22, 128.60, 126.24, 122.66, 111.50, 59.91, 59.06, 56, 44, 52.75, 50.92, 48.84.

Example 24 Preparation of 1- (2-methoxy-5-aminobenzyl) -1,4,7,10-tetraaza-20-cyclododecane-4,7,10-triacetic acid

To a nitrogen-purged aqueous solution (50 mL) containing 50 mg of PtO 2 was added 50 mg of 1- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10 -tri-acetic acid (prepared by the procedure of Example 23). After being hydrogenated in a Parr bomb for 1 hour, the solution was filtered and the aqueous filtrate freeze-dried to give the aniline derivative as a tan solid (95% yield), m.p. > 240 ° C (dec), and further characterized by the following data: 1 H-NMR (D 2 O): 7.54 (broad s), 7.21 (d), 7.09 (d), 7 , 05 (s), 6.88 (s), 3.84 (s), 3.78 (s), 2.85 - 3.56 (m); 13 C NMR (D 2 O): 180.59, 179.77, 153.38, 124.16, 124.03, 122.82, 120.18, 113.68, 112.43, 59.06, 57.02 , 55.96, 35 53.67, 50.52, 50.08, 49.70, 49.04, 48.32.

70 104898

Example 25 Preparation of 1- (2-hydroxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid

To an aqueous solution (1 mL) of 1- (2-hydroxy-5-5 nitrobenzyl) -1,4,7,10 -1-tetraazacyclododecane (200 mg, 0.62 mmol; prepared according to the procedure of Example F) was added bromoacetic acid. (309 mg, 2.2 mmol) and NaOH (2.5 mL, about 1 M) while stirring the solution at room temperature. The progress of the reaction was monitored by LC (anion exchange, Q-Sepharose ™), and the pH was maintained at approximately 8 by the addition of NaOH as needed. After 12 hours, the solution was lyophilized and the solid residue was chromatographed (column) on silica gel eluting with solvent mixture 5. The yellow major fraction was concentrated, dissolved in water and freeze-dried to give the desired product as a bright yellow powder (150 mg, 49%); ° C (decomposes) and was further characterized by the following data: 13 C NMR (D 2 O): 166.41, 165.60, 160.00, 119.91, 116.02, 113.85, 111.71, 104.59, 45.20, 44.50, 43.96, 36.43.

Example 26 Preparation of 1- (2-hydroxy-5-aminobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid: In an aqueous solution (25 ml) purged with argon, containing N- (2) -hydroxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (200 mg, 0.4 mmol; prepared by the procedure of Example 25), PtO 2 (130 mg) was added ) and subsequently passed with H2 (Parr hydrogenation device). After the yellow color had completely disappeared, the solution was purged with argon and filtered. The aqueous solution was then freeze-dried to give the desired product as a tan solid (146 mg, 78%).

35 71 104898

Example 27 α- [2- (4-Nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1- (R, S) -acetic acid-4,7,10-tris (R-methyl-ethyl) ) Preparation of 2-isopropyl-4,7,10-trimethyl ester of acid (PN-DOTMA-5 trimethyl isopropyl ester)

A solution of 1.00 g (2.37 mmol) of α- [3- (4-nitrophenyl) propyl] -1,4,7,10-tetraazacyclododecane-1-acetic acid 1-methyl ester (prepared according to Example P) procedure) in 35 mL of dry acetonitrile-10, 1.15 g (8.32 mmol) of anhydrous potassium carbonate was added while stirring vigorously under nitrogen. Optically active benzene sulfonate of the lactic acid methyl ester (1.79 g, 7.36 mmol, S: R = 98.2) was added and the mixture was stirred at room temperature for 60 hours. Analysis by TLC-15 showed the formation of new products (Rf = 0.71 for the title tetraesters, solvent system 2, and Rf = 0.89 for the triesters). The resulting solution containing the suspended carbonate salts was poured into 40 ml of chloroform and the precipitated inorganic salts were filtered off. Solvent was removed from this filtrate and the crude orange oil was chromatographed twice on a 2.5 cm x 41 cm flash silica gel column using solvent mixture 2 as the eluent to afford the title product as a whole; 25 (1.00 g, 1.47 mmol, 62% yield) which did not contain the alkylated products.

1 H NMR analysis of this material showed it to contain about 0.5 equivalents of unreacted benzenesulfonate-30 derivative. The NMR spectra of this material did not merge ··· at 6 ° C in chloroform: Ή NMR (CDCl 3, 60 ° C): 7.9 - 8.1 (m, 2H), 7.2 - 7.4 (m, 2H), 5.06 (m, 1H), 3.4-4.0 (m, 9H), 1.7-3.4 (m, 20H), 1.0-1. , 7 (m, 15H); 72 104898 IR (CDCl 3, cm -1): 2980, 2840, 1725, 1710 (estercarbonyl), 1590, 1510, 1440, 1340; FAB mass spectrum (m / e): 702 ([M + Na +] +), 687.

Example 28 5α- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1- (R, S) -acetic acid-4,7,10-tris (R- Preparation of methyl acetic acid 2-isopropyl-4,7,10-trimethyl ester (PA-DOTMA-trimethyl isopropyl ester) α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza -10-cyclododecane-1- (R, S) -acetic acid-4,7,10-tris (R-methylacetic) acid 2-isopropyl-4,7,10-trimethyl ester (950 mg, 1.40 mmol, uncorrected) (prepared by the procedure of Example 27) containing unreacted 1-methyl ester of α- [3- (4-nitrophenyl) propyl] -1,4,7,10-tetraazacacyclododecane-15-n-1-acetic acid was dissolved in 10 ml: 30% aqueous methanol containing 1.00 g of 10% Pd / C catalyst under a nitrogen atmosphere. Excess hydrogen was bubbled through the solution for 7 hours. At this point, TLC showed strongly formation of ninhydrin positive (Rf = 0.62, solvent mixture 2, starting material Rf = 0.71). The methanol-containing catalyst slurry was filtered through diatomaceous earth and evaporated to give 800 mg of the crude title product as a clear oil containing benzenesulfonic acid (Rf = 0.09) as a result of similar hydrogenolysis of the benzenesulfonate ester (Rf = 0.09). The crude oil was chromatographed on a 2.5 cm x 20 cm flash silica gel column using 10% methanol in chloroform as eluent to elute a 30-yellow pale yellow band in the pore space. Solvent mixture 2 was then used to elute the title product (480 mg) in 52% yield as a mixture of non-separated diastereomers and geometric isomers. In the aromatic region of the 1 H-NMR spectrum, three different AB quartets were observed: 73 104898 1 H-NMR (CDCl 3, 30 ° C): 6.63-8.0 (quartets, 4 H), 5.07 (tn, 1H, isopropyl methine-H), 3.53 - 3.9 (m, 13H, four singlets at 3.85, 3.73, 3.69 and 3.56), 3.46 (m, 1H), 3 , 25 (broad t, 3H), 2 to 3.1 (m, 14H), 1.0 to 2.0 (m, 15H); 13 C NMR (CDCl 3, 30 ° C): 177.3, 177, 176.4, 176.2, 175.9, 174.3, 172.9, 170.6, 145.3, 144.9, 130 , 8, 129.7, 129.3, 129.1, 128.8, 128.4, 127.5, 126.3, 115.2, 115.1, 114.9, 69.0, 68.4 , 67.3, 61.6, 57.8, 57.4, 56.7, 56.5, 56.4, 10, 52.9, 52.7, 52.1, 50.8, 50.7, 50.6, 47.3, 47.1, 47.0, 46.9, 46.5, 46.3, 44.8, 44.5, 44.3, 34.1, 32.9, 32.5, 32.2, 31.8, 24.8, 22.2, 22.1, 22.0, 21.8, 21.6, 15.8, 14.9, 7.7, 7.5, 7.4, 7.1; IR (CDCl 3, cm -1): 2960, 2840, 1720, 1510, 1450, 1375; FAB mass spectrum (m / e): 672 ([M + Na *] +), 686.

Example 29 α- (2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1- (R, S) -acetic acid-4,7,10-tris (R-methylacetic acid) ) acid (PADOTMA) Preparation of 20α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1- (R, S) -acetic acid-4,7,10- A mixture of diastereomers of 2-isopropyl-4,7,10-trimethyl ester of tris (R-methylacetic acid) (400 mg, 0.59 mmol; prepared according to the procedure of Example 28) was dissolved in 50 ml of concentrated N) hydrochloric acid, and the solution was refluxed for 30 hours.

TLC analysis (solvent mixture 1) showed that the ester (Rf = 0.98, solvent mixture 1) was transformed into two new spots (faster moving diastereomer Rf = 0.6 and slower moving Rf = 0.54, solvent mixture 1, both spots UV, ninhydrin and iodine positive). Excess solvent was removed on a rotary evaporator, and after drying, a mixture of crude diastereomers of the title product (403 mg) as the hydrochloride salt was obtained. Preparative separation of the two diastereomers of the title product was accomplished by transferring 290 mg (0.51 mmol) of the crude salt onto a 74 104898 flash silica gel chromatography column, 1 cm x 13 cm, eluting with solvent 8. Higher Rf diastereomer (119 mg, 0 , 21 mmol) was obtained in a purer form than the diastereomer (90 mg, δ 0.16 mmol) with a lower R

The Rf-diastereomer first eluted from the column. 1 H NMR spectrum of diastereomer with higher R f: 1 H NMR (D 20, 90 ° C, pD = 7.5): 7.12 (d, 2H), 6.80 (d, 2H) , 3.66 (m, 1H), 3.57 (broad t, 3H), 2-3.4 (m, 19-10H), 1.89 (m, 1H), 1.37 (m , 1H), 1.15 (d, 3H), 1.10 (d, 3H), 1.06 (d, 3H); 13 C NMR (D 2 O, 90 C, pD = 7.5): 184.4, 184.2, 184.0, 183.3, 135.9, 132.6, 131.7, 119.1, 78.2 , 69.0, 61.7, 61.5, 61.1, 57.4, 54.1, 49.7, 49.6, 48.2, 47.7, 47.2, 34.8, 33 , 8, 9.6, 15 9.1, 9.0; FAB Mass Spectrum (m / e): 566 ([M + H +] +), 588 ((M + Na +] +), 604 ([M + K +] +), 626 ([M + K * + Na + -). H *] +), 642 ([M + 2K + -H * n.

Preparation of the final products, complexes Metal ligand complexes were prepared by various methods as described below. The methods involved mixing the metal and ligand in an aqueous solution and adjusting the pH to the desired value. The complexing was carried out in solutions containing salts and / or buffers: 25 and water. At times, heated solutions were found to give better yields than complexing at ambient temperature. The product isolation and purification procedure used in the manufacture of BFC has also been shown to influence complex formation.

Example 30 Preparation of the samarium (III) complex [Sm (BA-DOTA)] of the ammonium salt of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [Sm (BA-DOTA)]

A small portion (53 mg, 0.094 mmol) of α- (4-aminophenyl) -35 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 75 104898 poa (prepared according to the procedure of Example 3) ) was dissolved in 100 μl. water and treated with 3 mL of 0.043 M Sm (OAc) 3 solution (48.8 mg, 0.128 mmol) at pH 6-7. This solution was heated at 100 ° C and the degree of complexation was determined by anion exchange HPLC. When the complex formation was complete according to anion exchange chromatography (HPLC system III), the solution was cooled to room temperature (about 25 ° C) and lyophilized. The samarium complex was purified by silica gel chromatography (solvent mixture 1 as eluent). This procedure yielded the title product in 82% yield. This complex was characterized by TLC, FAB mass spectrometry and anion exchange HPLC and further by the following data: 1 H-NMR (Da 0): 8.94, 7.81, 7.21, 6.97 , 6.80, 5.63, 5.01, 4.58, 4.01, 3.56, 1.78, 1.53, 1.24, 1.10, 0.77, -2.80, -2.95, -3.21, -3.91; 13 C NMR (D 6): 190.3, 184.9, 181.4, 146.8, 134.3, 122.7, 116.4, 80.8, 72.6, 64.2, 57.2 , 55.8, 54.7, 53.4, 51.5, 20 49.7, 45.5, 43.5, 23.6.

FAB mass spectrum: [M + H +] + = 645 (Sm isotope model). Example 31 The samarium (III) complex of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; 25 mists

A solution of 150 μΐ Sm-153 (3 x 10'4 mol / l in 0.1 N HCl) and 600 μΐ SmCl3 (3 x 10'4 mol / l in 0.1 N HCl) was prepared: ) by adding the Sm-153 solution to the SmCl3 solution. Then 5.0 μΐ of 2.0 M NaOAc was added followed by 45 μΐ of 50 mM ligand (prepared according to the procedure of Example 3) in HEPES buffer solution. The pH of the solution was adjusted to 275 μ1: 1ΐ3 0.5 M HEPES to 7. This solution was divided into two portions of 500 μ1: η and the other was heated for 1 hour at 100 ° C.

76 104858

The solutions were passed through a SP-Sephadex ™ cation exchange resin. The percentage of complexed metal was determined using the procedure described previously. The results show that 99.6% of the metal was complex 5 in the case of the heated sample and 96.6% in the case of the unheated sample.

PH stability of chelates

The stability of the metal-ligand complexes was measured by exposing them to various pH values and analyzing the complex from solution. At low pH, protonation of the ligand can cause metal release. At high pH, metal hydroxides compete with chelation. Thus, when searching for inert chelates, the desired inert chelate is thus retained as a chelate at both high and low pH.

The inertia profiles of some of the chelates obtainable according to the invention were formed using the methods described in the following examples. In some cases, the non-chelated metal was removed by passing the solution through a cation-20 exchange resin. Dilute sodium hydroxide and hydrochloric acid solutions were used to adjust the pH of the complex solutions from about 1 to about 14. The percentage of metal retained in the complex was then determined by the methods previously described.

: 25 In a similar manner, several bifunctional chelate conjugates were prepared and exposed to pH 2.8, 4.0 and 6.0. The amount of conjugate remaining in solution was determined by HPLC using a radiation measuring detector. The results are shown in the biological section in Example 30 cat XX.

·· Example 32 pH-stability of the samarium (III) complex of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 104898 77

The heated and unheated sample from Example 30 was each divided into two equal portions of 250 μΐ. The pH of the second batch of 250 μ1: η was adjusted with HCl to the values shown in the following table. The pH of the second batch of 250 μ1: η was adjusted to the desired values with microliter volumes of NaOH. When the desired pH was reached, a 25 μ1: η sample was taken and the percentage of the complexed metal determined as described previously. The results were a screening drab:

Complex% pH .Body- Heated uncommon 15 _______- Ί. 98.9 92.7 3__99 T 6 93f 3 5__99 T 6 92.9 7 99.6 95.7 20 9__99f 9 95 T 8 n__99.6 95.7 13__99.6 9H, 5. These results show the stability of this sample, whether heated or not, regardless of the test pH.

Example 33 Preparation of a Yttrium (III) Complex of α- (4-Aminophenyl) -1,4,7,10-tetraazacyclododecane-30 nα-1,4,7,10-tetraacetic acid in 10 µl of a solution containing 1 , 4 mg of α- (4-amino-phenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid / 100 μΐ (prepared by the procedure of Example 3), μΐ Y (OAc) 3 (0.003 M), 7β 104898 supplemented with Y-90. To this solution was added 500 μΐ of water followed by 125 μΐ of NaOAc (0.5 M). Water (265 μΐ) was added to the solution to bring the total volume to 1 ml. This solution was divided into two equal portions.

The second portion was heated to 100 ° C for 1 hour. Both heated and unheated sample were examined to determine the percentage of metal in the complex using the cation exchange procedure described above. The results showed that 84% of the metal was complex in the case of the hot sample and 4% in the case of the unheated sample.

Example 34 Preparation of the α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid lutetium (III) complex α- (4-aminophenyl) - A solution of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared according to the procedure of Example 3) was dissolved in distilled water to a concentration of 1.63 mg / ml.

A solution of lutetium chloride was prepared by dissolving lutetium chloride in 0.1 N HCl to give a solution of 3.9 mg / ml (0.01 M). Lutetium-177 (0.3 mM) was used as a marker.

30 μΐ of 0.01 M lutetium solution was added to 2 μΐ of Lu: 25 177 solution. An appropriate amount of the above ligand was added and added to 1.0 mL of HEPES buffer (0.1 M, pH 7.6) to increase the total volume. The resulting solutions were 0.3 mM for ligand and lutetium. The solution was then heated to 100 ° C for 1 hour, and the percent Lu of the complex was determined to be 86% by the previously described cation exchange method.

Example 35 Preparation of Samarium (III) -35 Compound o- (4-Isothiocyanatophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid 104898 79 Sample lot, 7 mg ( 10.8 μπιοί), α- (4-aminophenyl) -14.7.10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium (III) complex (prepared according to the procedure of Example 30) was dissolved in 400 μΐ. water.

An excess (50 μΐ) of thiophosgene was added followed by 400 μΐ of CHCl 3, and the biphasic reaction mixture was vigorously stirred for 30 minutes. The aqueous layer was then extracted with 500 μ1: 1-3 CHCl3 (Four times) and then lyophilized to give the desired title product in 10 quantitative yields.

In the UV spectrum, this compound exhibited an absorption peak at 272 nm and 282 nm. TLC (silica gel, developing with 75:25 by volume CH3CN-H2O) gave an Rf value of 0.38. The R £ value of the starting material is 0.19. The IR spectrum exhibited a SCN peak at 2100 cm -1; FAB Mass Spectrum: [M + H] * = 687.

Example 36 Preparation of the yttrium (III) -20 complex of the ammonium salt of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid Sample lot, 90 mg (160 mmol), α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared according to the procedure of Example 3) was dissolved in 1 ml of water. To this solution was added 3 ml: 25 water containing 51 mg (192 mmol) of Y (OAc) 3. The reaction mixture (pH = 6-7) was then heated at 100 ° C for 2 hours. The resulting solution was then passed through glass wool and freeze-dried to give 126 mg of a yellow solid. This solid was chromatographed on silica gel (solvent mixture 1) to give; 71 mg (76%) of the desired complex product. Anion exchange analysis showed a dwell time similar to that of the corresponding Sm complex. The title product was characterized by the following data: 80 104898 13 C NMR (D 2 O): 180.8, 180.5, 179.9, 146.1, 133.4, 122.0, 116.1, 74.9, 66 , 3, 56.3, 55.8, 55.4, 55.1, 54.8, 52.2, 46.0, 44.0, 23.6; 1 H-NMR (D 2 O); 6.90 (d), 6.70 (d), 4.40 (s), 3.45 - 3.06 δ (m), 2.71 - 2.10 (m), 1.75 (s) ; FAB Mass Spectrum: [M + H +] = 582.

Example 37 Preparation of Compound 10- Yttrium (III) Sodium Salt of α- (4-Isothiocyanatophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-Sample Lot o- (4-Aminophenyl) ) -1,4,7,10-Tetra-aza-cyclododecane-1,4,7,10-tetra-acetic acid yttrium (III) complex (prepared according to the procedure of Example 36; 10 mg, 17 μΐ) was dissolved in 400 μΐ. of water. To this 15 solution was added 64 μΐ (excess) thiophosgene and 400 μΐ CHCl 3 and the resulting reaction mixture was vigorously stirred for 40 minutes. During this time, several small additions of solid NaHCO 3 were made to maintain the pH at approximately 8. After completion of the reaction, the aqueous layer was separated and extracted with 1 mL of CHCl 3 (Fourth) and freeze-dried. The title product was characterized by TLC and UV spectroscopy.

Example 38 Ammonium salt of α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-aza: cyclododecane-1,4,7,10-tetraacetic acid yttrium (III) complex, NH 4 Preparation of [Y (PA-DOTA)]

Ligand, α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared according to the procedure of Example 8), converted to the protonated form and ammonium salt blend. sex chromatography using a strong cation exchange resin (SP-Sephadex ™ C-25, Pharmacia). The column was in protonated form and rinsed with distilled water. Li-gand concentrated aqueous solution was applied to the column, after which the column was rinsed with distilled water. The ligand was eluted from 104898 µl of the column with 0.5 M NH4OH. The eluent was removed by evaporation of the solution to dryness.

A solution of Y (OAc) 3 * 4H 2 O (0.0728 g, 0.215 mmol) in 5 mL of distilled water was added to a warm solution of a mixed ligand in protonated and ammonium salt (0.120 g, 0.215 mmol). ml of water. The solution was heated to reflux and adjusted to pH 7.0 with 0.1 M NH 4 OH. After one hour at reflux, the solution 10 was cooled and evaporated to dryness. Excess NH4OAc was removed by heating the white solid in an oil bath at about 105 ° C under reduced pressure. The yield of the title product (containing about 1 equivalent of ammonium acetate) was 0.142 g (94%) and was characterized by 13 C NMR (D 2 O, 88 ° C, 75 MHz): 23.8, 27, 6, 35.4, 49.6, 55.0, 56.2, 56.9, 57.1, 65.7, 68.0, 121.7, 132.6, 138.8, 180.7, 181.4, 182.0, 183.1 FAB mass spectrum (m / e): 610 (positive ion, [M '+ 20 2H +] +), 608 (negative ion, M').

Example 39 Ammonium salt of the samarium (III) complex of α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid, NH 4 [Sm Preparation of PA-DOTA)], · · * 25 By repeating the procedure of Example 38 using Sm (OAc) 3 * 3H 2 O (0.082 g, 0.215 mmol) in place of Y (OAc) 3 * 4H 2 O, the title compound was prepared. (containing about 1 equivalent of ammonium acetate) in a yield of 0.155 g (94%) and characterized by the following data: 13 C NMR (D 2 O, 88 ° C, 75 MHz): 23.5, 27, 2, 35.6, 50.5, 54.5, 4 56.0, 57.2, 57.9, 69.5, 71.5, 122.3, 133.0, 139.7, 181.2 , 188.4, 189.3, 190.9; 104898 82 FAB mass spectrum (m / e): 673 (positive ion, [M '+ 21Γ] +, Sm isotope model), 671 (negative ion, M', Sm isotope model).

Example 40 Ammonium salt of 5α- [2- (4-isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex, NH 4 [Sm (SCN) -PA-DOTA)]

Solutions containing Sm (OAc) 3-3H2O 10 (27.9 mg, 381.56 g / mol, 73.1 μπιοί) in 10 ml of distilled water and 42 mg (632.97 g / mol, 66 , 4 μπιοί) PA-DOTA Ammonium Potassium Mixed salt (prepared by the procedure of Example 8) in 10 ml of distilled water, stirred and heated on a steam bath. After 30 minutes, the reaction to form Sm (PA-DOTA) was complete by HPLC as determined by the procedure described in Example 41. The solution was reacted with another solution containing 45.4 mg CSCl2 (114.98 g / mol, 395 μπιοί) in 20 ml of chloroform by shaking them in a separatory funnel. The reaction was completed in about 1 minute as determined by HPLC and based on the absence of a positive reaction in the ninhydrin assay (0.2% in ethanol) when spot-analyzed on an silica gel TLC plate [starting chelate, Sm (PA-DOTA), was: · 25 in test positive]. The chloroform layer was removed and the aqueous layer was washed with two 20 mL portions of chloroform. The aqueous layer was evaporated to dryness by adding 100 ml of acetonitrile and evaporating at ambient temperature under a stream of nitrogen to give 56 mg of the title product.

FAB mass spectrum (m / e): 715 (positive ion, [M '+ 2 +] *, Sm isotope model), 713 (negative ion, M', Sm isotope model).

104898 83

Example 41 HPLC Analysis of Chelation Rate between Y3t and PA-DOTA The rate of chelation between PA-DOTA and Y3 + was investigated as a function of the product isolation and purification procedure used to prepare PA-DOTA. The degree of chelation formation was monitored by HPLC using a 100 mm Alltech Econosphere ™ C18 column. The gradient used was: A) 0.05 M NaOAc buffer, pH 6.0, and CH 3 CN in a ratio of 95: 5, B) 0.05 M NaOAc buffer, pH 6.0, and CH 3 CN in 30:70, A changed to B in 15 minutes. A solution of both PA-DOTA hydrochloride salt (residence time = 1.31 min) and PA-DOTA ammonium potassium mixed salt (residence time = 4.55 min), both prepared according to the procedure of Example 8, was prepared at a concentration of 1.6 mM in 0.5 M NaOAc buffer, pH 6.0. One milliliter (1.6 μπιοί) of each solution was reacted with 0.2 mL (8 μπιοί) of Y (OAc) 3 * 4H 2 O solution (40 mol / L in 0.5 M NaOAc buffer, pH 6.0). . Chelate formation was determined based on the peak at 20 retention time of 3.96 min (confirmed by comparison with the sample from Example 38). The results of chelation were as follows: 25 I PA-DOTA salt I Complex Time (min) / - You (%) temperature_.

> 90 line temperature.

PA-DOTA ammonium <10 15 / room temperature

potassium mixed salt> 85 10/90 ° C

· The method used to purify the BFC clearly affects the rate of chelation.

Example 42 α- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-35-cyclododecane-1- (R, S) -acetic acid-4,7,10-tris (methylacetic acid) - 104898 Preparation of the ammonium salt of the samarium (III) acid complex, NH [[Sm (PA-DOTMA)].

A solution of Sm (OAc) 3 * 3H 2 O (13.2 mg in 2 mL of distilled water) was added α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetra-5-azacyclododecane-1-one. Of a solution of (R, S) -acetic acid-4,7,10-tris (methyl acetic acid) (prepared according to the procedure of Example 29; higher diastereomer Rf) in a solution (30 mg in 2 ml of distilled water). The pH 6.5 solution was heated on a steam bath and the reaction monitored by 10 HPLC. After heating for 30 minutes, the solution was evaporated to dryness on a rotary evaporator and dried in a vacuum oven.

FAB mass spectrum (m / e): 715 (positive ion, [M + 2 H *] *, Sm isotope model), 737 (positive ion, [M + H + + Na *] *, 15 Sm isotope model), 713 (negative ion, M ', Sm isotope model).

In the following examples, solutions of the complexes were passed through an ion exchange column to remove any excess free metal from the solution. Labile systems-20 would return to equilibrium and have a similar amount of free metal in the solution. Inert systems would not return to equilibrium and the amount of non-chelated metal should not decrease. Thus, although the complex may be formed in low yield, purification by calculation: The ion exchange resin could yield a suitable stable complex which does not contain uncomplexed metal.

Example 43 Preparation of the samarium (III) complex of α- (2-methoxy-5-aminophenyl) -1,4,7,10-tetraaza-30-cyclododecane-1,4,7,10-tetraacetic acid

A solution of α- (2-methoxy-5-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared according to the procedure of Example 12) was prepared by dissolving 35 x 10'2 M. 5.8 mg of it to 325 μΐ of NANO-104898 85 pure ™ water. A 10 µl aliquot of this solution was added to 990 µl of 3 x 10 -4 M SmCl 3 (in 0.1 N HCl) supplemented with Sm-153. The pH of the solution was then adjusted to 7.0 with NaOH. This solution was divided into two 500 ml portions, one of which was heated for 1 hour at 100 ° C. Both the heated and the unheated sample were examined to determine the percentage of metal in the complex using the cation exchange procedure described above. The solution was then passed through a SP-Sephadex ™ resin. Again, complexation was determined from pure samples. The results are shown in the following table:

Heated Unheated 15 Purified Purified- Purified Uncleaned ton

Complete 96 95 97 89 you (%)

Example 44 pH-Stability of 20? - (2-Methoxy-5-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-Tetraacetic Acid Samarium (III)

The heated and unheated sample from Example 43 was each divided into two equal portions: * 25 portions of 250 µl. The pH of the second batch was adjusted with HCl and the second with NaOH to the values shown in the following table. Once the desired pH was reached, the sample was allowed to stand for 10 minutes and the percentage of complex metal previously determined by the cation exchange method was determined. The results are shown in the following table: 86 1 0 4 8 9 8

As a complex (%) pH RummyHeated madam 5 2 95 94 3 97 95 5__98 97 7__96 97 10 9__98 96 11__97 98 13 Ϊ55 99

Example 45 Preparation of 15α- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium (III) complex

Α- (2-Methoxy-5-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-20-p (prepared according to the procedure of Example 11) was prepared 3 x 10'2 M solution by dissolving 7.9 mg of it in 464 µl of NANO-pure ™ water. A 10 μl aliquot of this solution was added to 950 µl of 3 x 10 4 M SmCl 3 (in 0.1 N HCl) supplemented with Sm-153. The pH of the solution was then adjusted to pH 7.0 with NaOH; This solution was divided into two 500 ml portions, one of which was heated for 1 hour at 100. The sample, both heated and unheated, was assayed to determine the percentage of metal in the complex using the cation exchange procedure described above, and the solutions were run through an SP-Sephadex ™ resin. Complexation of the pure samples was then determined using the general procedure described above, and the results are shown in the following table: 87 104898

Heated Unheated 5 Purified Purified- Purified Uncleaned ton KömpTe ^ “100 72 100 46

Example 46 pH-stability of the 10? - (2-methoxy-5-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium (III) complex

The heated and unheated, purified sample from Example 45 was each divided into two equal portions. The pH of the second batch was adjusted with dilute HCl and the second with NaOH to the values shown in the following table. When the desired pH was reached, the sample was allowed to stand for 10 minutes and the percentage of complexing metal was determined by the previously described cation exchange method. The results are shown in the following table:

Refined 25 u ———.

• ph Room- Heated Infinite "" "2 99 ~ 99 Ϊ 100 100 30 5 Job Job 7 100 100 9 100 Job Job To Job 35 13 Job Job, 104898

Example 47 Preparation of the samarium (III) complex of α- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid α- (4-nitrophenyl) -1,4,7 A solution of 0.0219 M (100 μΐ, 2.195 mmol) of 10-Tetrazazacyclododecane-5-1, 1,4,7-triacetic acid (prepared by the procedure of Example 4) was contacted with 0.043 M Sm (OAc) 3 (25 mL). μΐ, 1.075 mmol). The reaction mixture was subjected to anion exchange-to-HPLC treatment (Q-Sepharose ™ column, 1 cm x 25 cm, 10 flow rate 2 mL / min, elution with 0-1 M NH 4 OAc gradient over 30 min). After 1 hour, the complex (dwell time - 11.1 min) was formed in 77% (area%) yield and was completely separated from the ligand peak (dwell time - 15.5 min). Further evidence of the formation of the title comp-15 complex was obtained by silica gel TLC (solvent mixture 4), with ligand Rf 0.59 and complex Rf 0.00.

Example 48 α- (4-Aminophenyl) -1,4,7,10-tetraazacyclododecane-20 nino-1,4,7-triacetic acid Samarium (III) Complex and α- (4-Aminophenyl) -1,4 Preparation of Samarium (III) Complex of 7,10-Tetrazazacyclododecane-1,4,10-Trietic Acid

A 0.022 M solution (100 μΐ, 2.2 pmol) of the isomers (prepared according to the procedure of Example 5) was contacted with a 0.043 M Sm (OAc) 3 solution (6.4 μΐ, 0.275 mmol). The reaction mixture was subjected to anion exchange-to-HPLC treatment (Q-Sepharose ™ column, 1 cm x 25 cm, flow rate 2 ml / min, elution with 0-0.25 M NH 4 OAc gradient over 30 min). After 40 minutes, the complex 30 (residence time = 9.0 min) was formed in 66% (area%) yield and was completely separated from the ligand peak • · (residence time = 18.5 min).

β9 104898

Example 49 Samarium-153 complex of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid and α- (4-aminophenyl) -1,4,7,10 Preparation of the samarium-153 complex of tetraaza-azacyclododecane-1,4,10-5 trietic acetic acid

Α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,7-triacetic acid pentahydrochloride and α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1-one were prepared. 3 x 10 ~ 2 M solution of 4,10-triacetic acid pentahydrochloride (prepared by the procedure of Example 10) by dissolving 4.2 mg in 300 µl of NANOpure ™ water. A 10 µl aliquot of this solution was added to 15 µl of 2 x 10 ~ 5 M SmCl3 (in 0.1 N HCl) supplemented with Sm-153. The solution was made up to 1 ml by addition of hydrogen. The pH was then adjusted to 7.0 with NaOH. This solution was divided into two 500 ml portions, one of which was heated at 100 ° C for 1 hour.

Both heated and unheated sample were assayed to determine the percentage of metal in the complex using the previously described cation exchange procedure. The results showed that 89% of the metal was complex in the case of the heated sample and 96% in the case of the unheated sample.

Example 50. '. 25 α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid samarium (III) complex and α- (4-aminophenyl) -1,4,7,10 pH stability of the tetra-azacyclododecane-1,4,10-triacetic acid samarium (III) complex

The heated and unheated sample from Example 49 was each divided into two equal portions. The pH of the second batch was adjusted with HCl and the second with NaOH to the values shown in the following table. Once the desired pH was reached, the sample was allowed to stand for 10 minutes and assayed for complexed metal.

Percentage of Iin by the cation exchange method described above. The results are shown in the following table:

As a complex (%) ^ pH of the body -. 'Heated unharmed 1 86 83 3 99 99 10 5 99 99 7 89 96 9 99 98 Γ 5 96 89 15 13 97 97

Example 51 Preparation of Samarium (III) Complex of α- (4-Nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid 20 A mixture of 13 μΐ α- (4- nitrophenyl) - 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (prepared according to the procedure of Example 4) 2 x 10'2 M solution, 15 μΐ 0.02 M SmCl3 (0.1 N in HCl) and 2 μΐ of Sm-153, a volume of 1 ml of NANOpure ™ water was made up. 25 adding. The pH was then adjusted to 7.0 with NaOH. This solution was divided into two 500 ml portions, one of which was heated at 100 ° C for 1 hour. Both the heated and the unheated sample were examined to determine the percentage of metal in the complex using the cation exchange procedure described above. The results showed that? 74% of the metal was in the case of the heated sample and 24% in the case of the unheated sample.

91 104898

Example 52 The pH stability of the Samarium (111) complex of 0- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid The heated sample of Example 51 was each divided into two for the most part. The pH of the second batch was adjusted with HCl and the second with NaOH to the values shown in the following table. Once the desired pH was reached, the sample was allowed to stand for 10 minutes and the percentage of complexing metal was determined by the cation exchange method described above. The results are shown in the following table: oH Complex (%) 15 1 * 67 2 70 5 70 ~ 5 72 20 7 7¾ 9 76 TO 76 12 "75 ... 25 I '3 I T6

Example 53 Preparation of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid lutetium (III) complex

A solution of EA-D03a (prepared by the procedure of Example 17 *) was prepared by dissolving the solid in water. The final concentration was 0.01 mmol / ml.

A lutetium chloride solution was prepared by dissolving 35 lutetium chloride in 0.1 N HCl. The resulting lutetium chloride concentration was 3.9 mg / ml (0.01 mmol Lu / ml). Lutetium-177 was used as a marker.

30 μΐ of 0.01 M Lu solution was added to 2 μΐ of Lu-177 solution. 30 μΐ of ligand was added and to increase the total volume to 1.0 ml of HEPES buffer (0.1 M, pH 7.6). The resulting solutions were 0.3 mM for ligand and lutetium.

The percentage of complex Lu was determined by the cation exchange method described above at 98%.

Example 54 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid Yttrium (III) Complex, Y (EA-D03A) Preparation 15 Ligand, 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (prepared according to the procedure of Example 17), was converted to the protonated form and the ammonium salt by chromatography using a strong cation exchange resin (SP-Sepha-20 dex ™ C-25, Pharmacia). The column was in protonated form and rinsed with distilled water. The concentrated aqueous ligand solution was applied to the column, after which the column was rinsed with distilled water. The ligand was eluted from the column with 0.5 M NH 4 OH. The eluate was removed by evaporation. The solution was dried on a rotary evaporator and dried in a vacuum oven.

A solution of Y (OAc) 3 * 4H 2 O (0.203 g, 338.101 g / mol, 0.600 mmol) in 10 mL of distilled water was added to a warm solution of mixed protonated and 30 ammonium salt (0.300 g, 499.615). g / mol, 0.600 mmol) in 10 mL of distilled water. The solution was heated to reflux and adjusted to pH 7.0 with 0.1 M NH 4 OH. After refluxing for 15 minutes, the solution was cooled and evaporated to dryness on a rotary evaporator. Excess 93 104898 ammonium acetate was removed by heating the white solid in an oil bath at about 105 ° C under reduced pressure. The yield of the title product (containing about 1 equivalent of ammonium acetate) was 373 mg (99%) and was characterized by 13 C NMR (D 2 O, 88 ° C, 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 mass spectrum (m / e): 552 (positive ion, [M + H +] *), 10610 (negative ion, [M + OAc ']').

Example 55 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid samarium (III) complex, Sm (EA-D03A) Preparation 15 By repeating the procedure of Example 54, using Y (OAc) 3 * 4H 2 O instead of Sm (OAc) 3 - 3H 2 O (0.229 g, 381.531 g / mmol, 0.600 mmol), the title product (containing ca. 1 equivalent of ammonium acetate) in 408 mg (99%) yield and characterized by the following data: 13 C NMR (D 2 O, 88 ° C, 75 MHz): 23.8, 27.0, 51.4, 57.5, 58.1 (3C), 68.7, 73.4, 120.3, 132.2, 134.7, 185.0, 186.8, 192.1; FAB mass spectrum (m / e): 615 (positive ion, [M + H +] +, 25 Sm isotope model), 673 (negative ion, [M + OAc "] ~,

Sm-isotooppimalli).

EXAMPLE 56 Preparation of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid samarium (III) carbamate ** '10 μΐ of a solution of 1.48 mg of 1- (4-nitrophenyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid / 100 μΐ (0.03 M; prepared in Example 20). ) was added to 15 µl of a 0.02 M SmCl3 solution 35 (in 0.1 N HCl) supplemented with Sm-153. The solution was made up to 1 ml by adding NANOpure ™ water. This solution was divided into two 500 ml portions, one of which was heated at 100 ° C for 1 hour. Both heated and unheated sample were assayed to determine the percentage of the complexed metal using the cation exchange procedure described above. The results showed that 81% of the metal was complex in the case of the heated sample and 43% in the case of the unheated sample.

Example 57 10 pH-stability of 1- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid samarium (III) complex

The heated sample of Example 56 was each divided into two equal portions. The pH of the second batch was adjusted with HCl and the second with NaOH to the values shown in the following table. Once the desired pH was reached, the sample was allowed to stand for 10 minutes and the percentage of complexing metal was determined by the cation exchange method described above. The results are shown in the following table: pH As a complex __L * l - _ 1 20 .. 25--: '2 37 4 49 5 58 7 81 30 9 62 ·· 10 64 12 64 il 54 35 95 104898

Example 58 Preparation of Samarium (III) Compound 1- [2- (4-Aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid 5 3 μΐ of a solution containing 4 , 65 mg of 1- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid / 100 ml (prepared according to the procedure of Example 20), 15 µl of & 3.η 0 02 M SmCl3 solution (0.1 N in HCl) previously Sm-153 added. The volume of solution 10 was made up to 1 ml by addition of NANOpure ™ water. This solution was divided into two equal portions, one of which was heated for 1 hour at 100 ° C. Both heated and unheated samples were assayed to determine the percentage of metal in the complex using the cation exchange procedure previously described. The results showed that 96% of the metal was complex in the case of the heated sample and 93% in the case of the unheated sample.

Example 59. PH stability of 1- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid samarium (III) complex

The heated and unheated sample of Example 58 was each divided into two equal portions. The pH of the second batch was adjusted with HCl and the second with NaOH to the values shown in the following table. Once the desired pH was reached, the sample was allowed to stand for 10 minutes and the percentage of complexing metal was determined by the cation exchange method described above. The results 30 are shown in the following table: * · «« · «96 104898

Complex (%) pH -

Heated Unheated 5 1 ~ MM 13 3 50 _M2 5 '73__55 7__96__93 10 9 9B 98

Tue 89 90 13 28 _37_

Example 60 Preparation of Samarium (li) complex of 1- (2-hydroxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid

A 3 x 10'2 M solution of 1- (2-methoxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid 20 (prepared by the procedure of Example 25) was prepared by dissolving 3, 9 mg of it in 260 ul of NANOpure ™ water. A 10 µl aliquot of this solution was added to 15 µl of 2 x 10'2 M aqueous SmCl3 supplemented with 10 µ 10 3 x 10 µM Sm-153 (in 0.1 N HCl). To 1 ml by addition of 965 μΐ distilled water, the pH was then adjusted to 7.0 with NaOH. This solution was divided into two 500 ml portions, one of which was heated at 100 ° C for 1 hour. to determine the percentage of metal in the complex 30 using the cation exchange procedure described previously .. The results showed that 71% of the metal was complex in the case of the heated sample and 74% in the case of the unheated sample.

97 1 0 4 8 9 8

Example 61 pH-stability of samarium (III) complex of 1- (2-hydroxy-5-nitrobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid 5 Heat and unheated sample according to Example 60 were each divided into two 250 μ1: η portions. The pH of the second batch was adjusted with HCl and the second with NaOH to the values shown in the following table. Once the desired pH was reached, the sample was allowed to stand for 10 minutes and 10% of the complexed metal was determined by the previously described cation exchange method. The results are shown in the following table:

Complex (%) 15 nH -

Pn Hot-.Heat Frozen 1 65 51 ~~ 3 * 98 93 ~ 20 5 "99 99 7 100 100 9 TOO 99 ΤΪ T5Ö 96 25 13 100 '98

Example 62 Preparation of 1- (2-hydroxy-5-aminobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid samarium (III) -30 Complex 1- (2-Hydroxy- 5-aminobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (prepared by the procedure of Example 26) 3 x 10'2 M solution by dissolving 3.2 mg in 100 µl of NANOpure ™ -35 water. A 10 µl aliquot of this solution was added to 15 µl of 2 x 98 104898 ΙΟ2 M aqueous SmCl3 supplemented with Sm-153. The volume of the solution was made up to 1 ml by the addition of 950 μΐ of distilled water and 20 μΐ of 0.1 N NaOH. This solution was divided into two 500 ml portions, one of which was heated for 1 hour at 100 ° C. Both the heated and unheated sample were examined to determine the percentage of metal in the complex using the cation exchange procedure described above. When less than 95% of the metal was complexed, the solution was passed through SP-Sephadex ™ resin 10. The percentage of complex metal was re-determined. The results are shown in the following table:

Heated Unheated 15 -—---- γ —-: -

Pure Pure Pure Purified Tummy Tummy Tummy

Complete 99 68 99 21 you (%) 20

Example 63 pH stability of the samarium (III) complex of 1- (2-hydroxy-5-aminobenzyl) -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid the unheated, purified sample was each divided into two 250 μl portions. The pH of one batch of 250 μ1: η was adjusted with HCl and the other with NaOH to the values shown in the following table. Once the desired pH was reached, the sample was allowed to stand for 10 minutes and the percentage of metal present as a complex determined by the cation exchange method described above. The results are shown in the following table: 99 104898

Complex (%) pH, Heated Heated 1 60 56 2 74 76 3 84 87 5 97 98 10 7 ~ 99 99 9 99 98 11 99 99 13 9999 15 Methods of Use - Biology

Bifunctional chelating agents containing a chelating moiety in the same molecule as a metal, such as DTPA, and a reactive linking group (arylamine) are known to be covalently linked to various target-targeting biomolecules that are specific for cancer or tumor cells. The radionuclide complexes contained in such conjugates are useful for diagnostic and / or therapeutic applications as a means of transporting the radionuclide to a tumor or cancer cell. [Reference: Meares et al., Anal. Biochem. 142: 68-78 (1984); see also the discussion in Meares and Goodwin, J. Protein Chem. 3 (1984), No. 2, pp. 215-15156; more recent literature include U.S. Patent 4,678,677 (Meares), issued July 7, 1987, U.S. Patent 4,652,519 (Warshawsky et al.), issued March 24, 1987, and Brechbiel et al., Inorg . Chem. 25, 2772-2781 (1986). It is contemplated that products using radioactive or loo 104898 luminescent metals may also be used in in vitro immunoassays.

background information

The usefulness of labeled antibodies will depend on many factors, for example (1) antibody specificity, (2) complex inertness or stability under conditions of use (i.e., stability in serum), and (3) antibody integrity, i.e., labeling process does not adversely affect antibody specificity or immune reactivity.

The stability or inertness of the complex is extremely important for the radionuclide-antibody conjugate to be effective as a diagnostic and / or therapeutic agent. Kinetic lability can lead to instability in pre-15 serum and release of the radionuclide from the complex. Thus, its diagnostic and therapeutic efficacy is reduced. In addition, the potential for general radiation damage to normal tissue increases. [Cole et al., J. Nucl. Med. 28: 83-90 (1987).] 20 Addition of a radionuclide to an antibody

The incorporation of the radionuclide into the antibody can be accomplished by conjugating the antibody to a preformed (ambient or elevated temperature) metal BFC complex. [Meares et. al., Acc. Chem. Res. 17, 25, 202-209 (1984).] Examples are given:

Example ZA (Comparative Example) Conjugation of 1- (4-isothiocyanate-benzyl) -diethylenetriamine-pentaacetic acid samarium-153 complex to IgG and F (ab ') 2 of CC-49; [153Sm (SCN-Bz-DTPA)] -IgG and 30 [153Sm (SCN-Bz-DTPA)] -F (ab ') 2 fragment: 1- (4-Isothiocyanate-benzyl) -diethylenetriaminepentaacetic acid samarium-153 complex [ 153Sm (SCN-Bz-DTPA)] was prepared by mixing with 150 µl of a solution of 153Sm in 0.1 N HCl, 9 μΐ of 1- (4-isothiocyanate-benz-35-yl) -diethylenetriaminepentaacetic acid. 104898 adjusted HEPES buffer (0.5 M, pH 8.9, about 30 μΐ) to adjust its pH to approximately 6. To effect conjugation, 153.5mg of IgG or CC-49 was mixed with the 153Sm complex. of F (ab ') 2 (protein concentration about 5 1 x 10 4 4 mol / L in 50 mmol HEPES, pH 8.5) and the pH was adjusted with sodium carbonate solution (1.0 M, 12- 15 μΐ) to 8.9 Conjugation was carried out at room temperature (about 25 ° C) for about 3 hours, IgG or F (ab ') 2 labeled with 153Sm complex was isolated and Karak-10 was sterilized as described in Example XII.

Experimental part on biology

Examples I and II and Comparative Examples A to D:

In vivo study of bifunctional chelates

The stability of some rare-earth metal chelates has been tested in An vivo animals. For example, Rosoff et al. have reported in International Journal of Applied Radiation and Isotopes 14, 129-135 (1963) the distribution of radioactive rare earth chelates in mice to certain aminocarboxylic acids. It was found that in vivo "competition between the chelating agent and constituents (inorganic and organic) of the rare earth ions determines its deposition and secretion". The strong earth chelates of the rare earth metals of this invention are expected to be degraded to a very small extent and excreted, whereas the weak and medium-strength chelates known in the art are more readily degraded and thus deposited in organs such as the liver. However, liver radionuclide content is not always due to the formation of a weak complex, but is in some cases the result of ·; the affinity of the metal chelate for the liver. In fact, chelates have been prepared and used to study liver function (Alan R. Fritzberg, Radiopharmaceuticals: Progress and Clinical Perspectives, Vol. 1, 1986; U.S. Patent Nos. 4,088,747 and 4,091,088).

102 104898

The biodistribution of the yttrium and samarium chelates of the compound of Example 3, which are examples of strong chelates of rare earth metals, was determined and a percentage of the dose was used as a qualitative evaluation of chelate stability in an in vivo assay. NTA and EDTA chelates are included for comparison purposes. Samarium was also injected as a reference substance in unchelated form as samarium chloride.

10 Sprague-Dawley rats weighing 150-200 g were purchased from Charles River Laboratories. These animals were housed in cages and given unlimited water and food. Animals were acclimatized to the conditions for at least 5 days prior to use. Prior to the injection of complex 15, the animals were placed under a heat lamp (15-30 minutes) to dilate the tail vein. The animal was then placed in a confining cage, wiped with alcohol, and injected (50-200 μΐ) into the tail vein. After injection 20, the animal was placed in a second cage for 2 hours, after which it was sacrificed by neck dissection. The animal was then dissected, rinsed with deionized water, flushed dry and weighed into a weighing flask. At least 3 control samples of the same substance were injected and counted with animal tissues. The percentage of dose is the number of pulses in the organ divided by the number of pulses in the control sample multiplied by 100 (see table below).

30 «· 103 104898

Table

Biodistribution Data '': jThe liver 5 Example Ligand pre- Metal blank (% h no sign * of injected dose Ϊ 3 Ϊ 0.17 II 3 Sm 0.29

10 (A) ~ EDTA Sm__S.H

(B) EDTA Sm (cl NTA Sm 8.6 (D) SmC1.3 Sm 39 15) The ligand-metal ratio of 1: 1 in Examples I and II, 5: 1 in Examples A and about 300: 1 in Examples 5 and C was used to prepare the complexes. .

Examples III and E

By the methods described above, 1: 1 complexes of Yttrium 20 (to which a small amount of 90Y was added) were prepared with the ligand of Example 3, BA-DOTA, and EDTA (Comparative Example E). A few aliquots of 100 µl were then transferred to a separate centrifuge tube. Y (III) was added in excess so that the total volume change was obtained. 25 was minimized and time was recorded. After half an hour, the amount of addition of the metal complex as a percentage as described above kationinvaihtomenetelmäl-la and compared to the original amount of complex. The percentage of complex as a function of added metal provides an indication of the lability of the ligand-metal complex. .. The results are shown in the table.

104 104898

Table

Metal and Li- As a Complex (%) 5 gand mole __

Ratio 3A-D0TA EDTA

Ϊ 80 98 10 - 86 10 100 - 78 250 88 H8 500 80 16

Example IV

1- [2- (4-Isothiocyanatophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid samarium-153 complex, [153Sm (SCN-EA-D03A)] - preparation of the complex

To 100 μl. solution containing 153Sm 0.1 N

In HCl, 5 μΐ SCN-EA-D03A (5 mmol / l in 50 mM 20 HEPES, pH 8.2) was added. This mixture was stirred in a vortex mixer and HEPES buffer (0.5 M, pH 8.9; total about 25 μΐ) was gradually added to adjust the pH to approximately 7. Chelation progress was monitored by HPLC using a GF-250 column. (HPLC sys-: Theme I). A yield of about 50% was obtained.

Example V

tt- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid samarium-153-

Preparation of Complex, [153Sm (PA-DOTA)] Complex, 30 150 μl of a solution containing 153Sm in 0.1 N

In HCl (about 4.6 mCi), 1 μΐ sodium acetate (2.0 M) and 9 μΐ α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1 were added. Ammonium potassium mixed salt of 4,7,19-tetraacetic acid (5 mmol in 50 mmol HEPES, pH 35 8.5, prepared according to the procedure of Example 9).

105 1 0 4 8 9 8

The mixture was mechanically stirred and titrated gradually with HEPES buffer (0.5 M, pH 8.9, about 31 µl) to pH 7. This mixture was then heated at 98 ° C in a particle bath for 1 hour. At the end of heating, 5 μΐ of Mixture-5 was used for analysis on a Mono-Q ™ column using HPLC system II, eluting with a gradient solvent system (0-15 minutes, 0% to 100% B; where A = water) and B = 1.0 M ammonium acetate containing 0.1 mmol EDTA). 85-95% yield based on 10 and 153Sm was achieved. The samarium-153 complex of o- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid thus prepared was characterized by comparing its chromatographic behavior with Mono-Q. ™ and GF-250 column for identical non-radioactive sample 15 behavior.

Further evidence of the presence of the radioactive complex was obtained by converting it to an isothiocyanate derivative and then conjugating it to the antibody. The complex is also prepared without heating (at ambient temperature) for 6-18 hours incubation, resulting in a 70-80% yield.

Example VI

α- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid identical to 253-153 complex, [153Sm (SCN-PA -DOTA)] complex

To the reaction mixture obtained in Example V, 2 μΐ HEPES buffer (0.5 M, pH 8.9), 2 μΐ thiophosgene and 0.2 ml chloroform were added. The mixture was vigorously stirred me-30 2 or 3 times for a few seconds at a time. Cl *; the roform layer was discarded and the aqueous layer containing mainly the desired product was saved and further purified. The yield of the samarium-153 complex of α- [2- (4-isothiocyanatophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-35-one was by HPLC GF-250 - 106 to 104898 columns based on 153Sm activity measurement using HPLC system I 85-90%. The aqueous layer was passed through a SEP-Pak ™ C-18 cartridge for purification and eluted with an acetonitrile-water mixture containing 90% 5 acetonitrile. The first 300 μΐ of eluate was discarded, and the subsequent 900 μ ulos of the SCN derivative was characterized by HPLC on a GF-250 column. Recovery of 153Sm activity was better than 90%. Most of the solvent was then evaporated over 1.5 to 2 hours and the residue used for conjugation to the antibody.

Example VII

Preparation of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex, 15 [153Sm (BA-DOTA)] complex

200 µl of a solution containing 153 Sm 0.1 N

In HCl, 1 μΐ of sodium acetate (2.0 M) and 12 μΐ of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,19-tetraacetic acid (5 mmol) were added. in mmol HEPES, pH 8.5 (prepared by the method of Example 3). The mixture was mechanically stirred and titrated gradually with HEPES buffer (0.5 M, pH 8.9, approximately 38 μΐ) to pH 7. This mixture was then heated at 98 ° C in a particle bath for 1 hour. After heating, 5 μΐ of the mixture was used for analysis on a Mono-Q ™ column (HPLC system II, gradient solvent elution: 0-15 minutes, 0% to 100% B, where A = water) and B = 1.0 M ammonium acetate containing 0.1 mmol EDTA). Generally, 85-95% yields based on 153Sm were achieved. The complex is also prepared by incubation ·; mixture at room temperature for 12-18 hours resulting in 80-90% yield of the title product. The samarium-153 complex of α- (4-aminophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 35 thus prepared was characterized by comparing its chromatographic behavior from Mono-Q ™. column to convert the authentic sample to its isothiocyanate derivative and then conjugate it to the antibody.

Example VIII

5α- (4-Isothiocyanate-phenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex, [153Sm (SCN-BA-DOTA)] complex , manufacture

To the reaction mixture obtained in Example VII was added 2 μΐ HEPES buffer (0.5 M, pH 8.9), 2 μΐ thiophosgene and 10 0.2 ml chloroform. The mixture was vigorously stirred mechanically 2 or 3 times for a few seconds at a time. The chloroform layer was discarded and the aqueous layer containing mainly the desired product was spared and further purified, the same as α- (4-isothiocyanatophenyl) -1,4,7,10-15 tetraazacyclododecane-1,4,7,10-tetraacetic acid The yield of the 153 complex was greater than 90% by HPLC on a GF-250 column using HPLC system I based on 153Sm activity. The aqueous layer was passed through a SEP-Pak ™ C-18 cartridge for purification and eluted with an acetonitrile-water mixture containing 90% acetonitrile. The first 0.3 mL of eluate was discarded, and the desired product came out in the next 1.2 mL in a 86-93% yield. Most of the solvent was then evaporated for about 2 hours and the residue was used in an · 25 conjugations.

Example IX

Conjugating the samarium-153 complex of 1- [2- (4-isothiocyanatophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid to the antibody; [153Sm (SCN-EA-30 D03A)] - IgG Conjugate ·; · The antibody used was the epitope of CC-49, a murine monoclonal IgG that binds TAG-72, a tumor-associated antigen. To effect conjugation, 187 μΐ antibody solution (1.20 x 10'4 mol / l in 50 mM 35 HEPES, pH 8.5) was mixed with 4.5 x 10 x 8 mol 153Sm (SCN-EA-10910). 4,8 9,8 D03A) prepared as described in Example IV and then sodium carbonate solution (1.0 M) was added to the mixture to raise its pH to 8.9. The reaction was allowed to proceed for 2 hours at room temperature.

Upon completion, 153Sm-labeled IgG was isolated by centrifugal gel filtration in Sephadex GF-25 (2.2 mL) disposable columns and further purified by HPLC on a GF-250 column using citrate buffer (0.25 M, pH 7 as eluent). , 4). The fractions containing labeled IgG were combined and concentrated and exchanged (3x) with PBS using Centricon ™ concentrators. The 1S3Sm-labeled IgG so prepared had a specific activity of about 0.16 μCi / μg protein. The integrity of [153Sm (SCN-EA-D03A)] -IgG was confirmed by HPLC [S. I. Sivakoff, BioChrom 1 15 (1986), No. 1, 42-48] and by conventional biochemical procedures, for example, sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography, and radioimmuno-solid phase assay (RIA). [See. David Colcher et al., Cancer Res. 43: 20736-742 (1983).]

Example X.

Conjugation of the samarium-153 complex of 1- [2- (4-isothiocyanato-phenyl) ethyl] -1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid to F (ab ') 2- of CC-49 fragment; 25 [153Sm (SCN-EA-D03A)] - CC-49-F (ab ') 2 fragment

To the F (ab ') 2-fragment of CC-49 prepared by enzymatic digestion according to the procedure described by Lamoyin and Nisonoff [E. Lamoyi and A. Nisonoff, J. Immunol. Methods 56: 235-243 (1983) (225 μΐ 1 × 10′4 M)

30 solutions of 50 mM HEPES, pH 8.5) were mixed with 2.9 x 10 -8 mol of 1S3Sm (SCN-EA-D03A) prepared as described in Example IV. Sodium carbonate (1.0 M, about 5 μΐ) was added to adjust the pH to 8.9, and the reaction was continued for about 2.5 hours. Upon completion, 109-104898 153Sm-labeled fragment was isolated and characterized as described in Example IX.

Example XI In vivo localization of [153Sm (EA-D03A)] -IgG and [153Sm (EA-D03A)] -F (ab ') 2 with t153Sm (EA-D03A)] the utility of labeled CC-49 IgG (from Example IX) and F (ab ') 2 (from Example X) was demonstrated by the accumulation of labeled materials in human tumorous graft of nude mice. Female thymus mice (Nu / Nu, CD1 background) were inoculated subcutaneously (s.c.) (0.1 ml / source) in a human colon cancer cell line, LS-174T (about 1 x 10 6 cells / animal). Approximately 2 weeks after inoculation, each animal was injected via tail vein 15 with approximately 2 μ (Γϊ (15-30 μg)) of 153Sm-labeled antibody in PBS. Mice were sacrificed at various intervals. After bleeding, tumor and certain tissues were excised and and their radioactivity was measured with a gamma counter. The number of pulses per minute (min "1) caused by 153Sm in each tissue was determined and expressed as min'Vgram of tissue per injection dose multiplied by 100 (% of injected dose / gram). are shown in Figures 1-14 and Tables IA and IB. IgG j '25 labeled with 153Sm (SCN-Bz-DTPA) and F (ab') 2 of CC-49 (from Example ZA) were included in the study. The results are shown in Tables IC and ID.

Example XII

conjugation of α- [2- (4-isothiocyanatophenyl) ethyl] -1,4,7,10-30 tetraazacyclododecane-1,4,7,10-tetraacetic acid complex to the antibody; [153Sm (SCN-PA-DOTA)] - IgG Conjugate The antibody used was the epitope of CC-49, a murine monoclonal IgG that binds to TAG-72, an antigen associated with tumors. To perform conjugation, 110 104898 sex of 178 μΐ antibody solution (1.26 x 10 4 mol / l in 50 mM HEPES, pH 8.5) was mixed with 3.4 x 10 ~ 8 mol a- [2- ( 4-Isothiocyanate-phenyl) -ethyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex 5 (prepared as described in Example VI) and added to the mixture. sodium carbonate solution (1.0 M, about 17 μΐ) to raise its pH to 8.9. The reaction was allowed to proceed for 2 hours at room temperature. Upon completion, 153Sm-labeled IgG was isolated by centrifugal gel filtration on Sephadex ™ GF-25 (2.2 mL) disposable columns and further purified by HPLC on a GF-250 column using citrate buffer (0.25 M, pH 7, eluent) as the eluent. 4). Fractions containing labeled IgG were pooled and concentrated and exchanged (3x) with PBS using Centricon ™ -15 concentrators. The 153Sm-labeled IgG so prepared had a specific activity of about 0.16 μCi / μg protein. The homogeneity and integrity of the samarium-153 complex IgG preparation of α- [2- (4-isothiocyanatophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid was confirmed by HPLC: and standard biochemical procedures as in Example IX.

Example XIII

Conjugation of α- [2- (4-Isothiocyanate-Phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid Compound 153 of CC-49 (ab ') 2 fragment; [153Sm (SCN-PA-DOTA)] - CC-49-F (ab ') 2 -f fragment to F-ab' 2 'fragment of CC-49 [prepared by the enzymatic degradation procedure of E. Lamoyi et al., J. Immunol. Methods 56, 30 235-243 (1983) (225 μΐ 1x ΙΟ 4 M solution containing 50 mmol HEPES, pH 8.5) was mixed with 2.9 x 10 "8 mol as described in Example VI. Prepared α- [2- (4-iso-thiocyanate-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153. Compound sodium carbonate (1.0) was added. M, about 9 μΐ) to adjust the pH to 104898 to approximately 8.9, and the reaction was continued for about 2.5 hours The 153Sm-labeled fragment was isolated and characterized as described in Example IX. / μ%.

Example XIV (A and B) N - [2- (4-Isothiocyanatophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid labeled with the samium-153 complex IgG or F (ab ') 2 conjugate, [153Sm (SCN-PA-DOTA)] -IgG and [153Sm (SCN-10 PA-DOTA)] -F (ab1) 2, localization in vivo of IgG of CC-49 labeled with α- [2- (4-isothiocyanatophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex: The utility of n or F (ab ') 2 (from Examples XII and XIII) was demonstrated by the accumulation of labeled material in human tumorous graft of nude mice. The biodistribution was determined by the method described in Example XI. The results are shown in Figures 1-7 for IgG and Figures 8-14 for F (ab ') 2; see. See also Tables IIA and IIB.

153Sm (SCN-Bz-DTPA) -labeled IgG and CC-49 F (ab ') 2 (prepared in Example ZA) were included in the study for comparison. [See. Tables IC and ID.] Example XV

: Conjugation of 25 α- (4-isothiocyanatophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex to antibody; [153Sm (SCN-BA-DOTA)] - IgG Conjugate to Total IgG of CC-49 (174 μΐ 1.2 x 10'4 mol / L in 0.25 M HEPES, pH 8.7) 2.0 x 10'8 mol of α- (4- Γ * isothiocyanate-phenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex (2.8 mCi ) prepared according to Example VIII and then sodium carbonate solution (35 M, 1.0 M, about 2 μΐ) was then added to the mixture to maintain its pH at about 112 104898 at 8.7. The reaction was allowed to proceed for 2.5 hours at room temperature. Upon completion, 153Sm-labeled IgG was isolated by centrifugal filtration on Sephadex ™ GF-25 (2.2 mL) disposable columns and further purified by HPLC on a 5 GF-250 column using citrate buffer (0.25 M, pH 7.4) as eluent. Fractions containing labeled IgG were pooled and concentrated and exchanged (three times) with PBS using Centricon ™ concentrators. The homogeneity and integrity of the IgG preparation labeled with ls3Sm were confirmed by HPLC and standard biochemical procedures as set forth in Example IX.

Example XVI

Conjugation of the α- (4-isothiocyanate-phenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid samarium-153-15 complex to the F (ab1) 2 fragment of CC-49 ; [153Sm (SCN-BA-DOTA)] - CC-49-F (ab ') 2 fragment to CC-49 F (ab') 2 -f fragment (84 μΐ 2.4 x 10'4 M solution, with 0.25 M HEPES buffer, pH 8.7) prepared by enzymatic digestion according to the procedure of E. Lamoyi et al., mixed with 2.1 x 10 ~ B mol a- (4 -isothiocyanatophenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid samarium-153 complex (prepared according to the procedure of Example VIII). Sodium carbo- was added; 25 N (1.0 M, about 2 μΐ) to adjust the pH to approximately 8.7, and the reaction was continued for approximately 2 hours. Upon completion, the 153Sm-labeled fragment was isolated and characterized as described in Example IX.

Example XVII (A and B) IgG of 30α- (4-Isothiocyanato-phenyl) -1,4,7,10-tetraaza-aza-cyclododecane-1,4,7,10-tetraacetic acid labeled with samarium-153 (Example XV) and F (ab ') 2 (Example XVI), [153Sm (BA-DOTA)] -IgG and [153Sm (BA-DOTA)] -F (ab') 2 in vivo 113 104898

In vivo studies were performed as described in Example XI and the results are shown in Figures 1-14 and Tables IIIA and IIIB.

Example XVIII (A and B) 5α- [2- (4-Isothiocyanatophenyl) ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 177Lu complex-labeled IgG and F (ab ') 2, [177Lu (PA-DOTA)] -

In vivo localization of IgG and [177Lu (PA-DOTA)] -F (ab1) 2 The title compounds were prepared and in vivo assays were performed as described in Examples V, VI, XI, XII and XIII, and the results are shown in Tables IVA and IVB.

Example XIX (A and B) IgG Labeled with 15α- [2- (4-Isothiocyanato-phenyl) -ethyl] -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid Yttrium-90 Complex: in vivo localization of n and F (ab ') 2, [90Y- (PA-DOTA)] -IgG and [90Y (PA-DOTA)] -F (ab *) 2 were prepared and in vivo studies were performed as described in Example XI except that the tissues were disrupted and subjected to liquid scintillation counting, and the results are shown in Tables VA and VB.

: 25 Example XX

In vitro pH stability of [153Sm (BFC)] -CC-49-IgG and [177Lu (BFC)] -CC-49-IgG or [153Sm (BFC)] -CC-49-IgG or [ 177Lu (BFC)] -CC-49-IgG was allowed to stand in 0.2 M NaOAc buffer at pH 6.0, 4.0 and 2.8 at room temperature with a protein concentration of about Γ5x10 106 mol / l and with the complex and the antibody being about 0.5. Samples were taken at specific time intervals and analyzed by HPLC (GF-250 column) to determine the loss of 153Sm or 177Lu activity from the protein. The study generally lasted 5 days or until 104898 90% of the radioisotopes were dissociated. The results are expressed as the initial daily rate of radioisotopic loss. The results showed 5 excellent values for 153Sm (PA-DOTA), 177Lu (PA-DOTA), 153Sm (BA-DOTA), 153Sm (PA-DOTMA) and 153Sm (MeO-BA-DOTA). stability in the acidic pH range with reference complex 153Sm (Bz-DTPA). The results are shown in the table below.

This greater stability of the complexes in vitro also correlates well with the faster elimination of 153Sm and 177Lu from the body 10 and non-target tissues (e.g., kidney, liver); see. Figures 1-34.

Isotope loss (%) / day SPC Isotope pH

15 6.0 V 2? 8

Bz-DTPA 'IDJSm <2 35 35 PA-DOTA' ^ Sm <2 <2 <2 BA-DOTA 1 ^ Sm <2 <2 <2 PA-DOTA ^ '<Lu <2 <2 <2 20 j - "—---------------

MeOBA-DOTA 1:? JSm <2 <2 <2 EA-D03A ^ Sm <2 90 95 PA-DOTMA 153sm <2 <2 -; Example XXI IgG Labeled with Samarium-153 Complex of α- (2-Methoxy-5-aminophenyl) -1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid and F (ab ') 2, [153Sm (MeO-BA-DOTA)] -IgG and [153Sm (MeO-BA-DOTA)] -F (ab1) 2, in vivo; * The title compounds were prepared according to the procedures set forth in Examples V, VI, XII and XIII, and in vivo studies were performed according to the procedures of Example XI. The results are shown in Tables VIA and VIB.

35 115 104898

Example XXII

To 100 μΐ of 153Sm (0.3 x 1033 M in 0.01 N HCl; 5 mCi) was added 6 μΐ of PA-DOTMA (diastereomer 5 prepared by the method of Example 29 having high Rf; 5 mmol / L in Milli-Q ™ water), 20 μΐ MES buffer (1.0 M, pH 6) and 1 μΐ HEPES buffer (0.5 M, pH 8.7). This solution was stirred on a vortex mixer and had a final pH of about 6. After heating at 90 ° C for 30 minutes, it was analyzed by 10 HPLC on a GF-250 column for degree of complexation, and generally achieved at least 90%: n yield. 153Sm-DOTMA was characterized by comparison with a non-radioactive Sm complex prepared and characterized separately as described in Example 42.

Example XXIII

Preparation of 153Sm (SCN-PA-DOTMA)

To the 153Sm (PA-DOTMA) complex prepared in Example XXI, cooled at room temperature for 20 minutes, was added 10 μΐ of a 1% thiophosgene solution if the medium was an acetonitrile / water mixture containing 90% acetonitrile. The mixture was vigorously stirred in a vortex mixer and allowed to stand at room temperature for 5-20 minutes. According to HPLC analytical monitoring, the reaction occurred immediately. The activated 153Sm complex • '25 was extracted three times with chloroform (200 μΐ / time) and the aqueous layer passed through a PRP cartridge (PRP cartridge = Mini-clean ™ cartridge purchased from Alltech Associates, Deerfield, IL) was pretreated with 5 mL of methanol and 5 mL of MES buffer 30 (20 mM, pH 5.8). After washing with 5 ml of MES-T buffer and 5 ml of Milli-Q ™ water, it was extracted with 900 μ1: 1-3% 90% acetonitrile, discarding the first 100 μΐ, yielding about 90% 153Sm -aktii-isotype. The mixture was evaporated to dryness under reduced pressure at a temperature below 40 ° C and the residue contained most of the title compound which was used for conjugation of the antibody.

Example XXIV (A and B) Conjugation of 153Sm (SCN-PA-DOTMA) to IgG and F (ab1) 2-5 to CC-49

Generally, the antibody was concentrated and exchanged with carbonate buffer (pH 9.1 or 9.5, 50 mM) in a Centricon ™ concentrator (smaller than 30 kilodaltons), resulting in at least a pro-10 protein concentration of 1.5 x 10 -4 mol. / 1. To effect conjugation to the 153Sm (PA-DOTMA) isothiocyanate derivative (prepared in Example XXIII), a small amount of carbonate buffer was required to reduce the final protein concentration to 1.5 x 10 -4 moles / L, followed by an equivalent amount of concentrated antibody relative to BFC-153 Sm-complex. The mixture was stirred in a vortex mixer and allowed to react at room temperature for 1 hour or until 40-50% of the 153Sm was bound to the antibody, according to HPLC analysis on a GF-250 column. The labeled antibody was isolated by two successive centrifugal filtration using Sepha-dex ™ G-25 columns (2.2 mL). The homogeneity, integrity and immunoreactivity of the labeled antibody were determined by HPLC analysis and standard biochemical procedures described above.

Example XXV (A and B) Biodistribution Studies on 153Sm (PA-DOTMA) Labeled IgG and F (ab ') 2-CC-4 9

Studies were performed as described in Example XI. The results are shown in Tables VIIA and VIIB. See. pictures 15 - 28.

Example XXVI

Preparation of 177Lu (PA-DOTMA) Complex 30 µl: 33η 177Lu solution (6 x 10 103 mol / l in 1.1 N 35 HCl, 4 mCi) was added to 36 µΐ of PA-DOTMA (prepared according to Example 117104898). 29) in 5 mM Milli-Q ™ water. The solution was stirred in a vortex mixer, and 115 μΐ MES buffer (1.0 M, pH 6.0) was added to adjust the pH of the solution to approximately 5.5 to 6. This solution was passed at 90 ° C for 30 minutes over 90 - to achieve a% chelation. The mixture was passed through a PRP cartridge pretreated with 5 mL of methanol and 5 mL of MES buffer (20 mM, pH 5.8). It was washed with 5 ml Milli-Q ™ water. The complex obtained with 1000 µl of 90% acetonitrile extracted corresponded to about 78% of the initial 177Lu activity (the first 100 µΐ of the extract was discarded).

Example XXVII Preparation of 177Lu (SCN-PA-DOTMA) in 90% Acetonitrile 177Lu (PA-15 DOTMA) Complex obtained in Example XXVI was added 6 μΐ of a solution containing 10% thiophosgene and 90% acetonitrile. This solution was stirred in a vortex mixer and after 20 minutes it was analyzed for isothiocyanate derivative formation by HPLC. The reaction is usually quantitative. Removal of the solvent and excess thio-phosgene was effected by evaporation under vacuum for 1-2 hours at a temperature below 40 ° C. The residue was used for conjugation of the antibody.

Example XXVIII

Conjugation of 177Lu (PA-DOTMA) to IgG-CC-49

An IgG-CC-49 antibody in carbonate buffer (50 mM, pH 9.1) was mixed with an equivalent molar mass of the isothiocyanate derivative in the buffer (prepared by the procedure of Example XXVII). The reaction was continued for 70 minutes at an antibody concentration of 1.5 x 10 4 M, and the labeled antibody was isolated and characterized according to the procedure described in Example IX.

118 104898

Example XXIX

Studies on the long-term biodistribution of 177Lu (PA-DOTMA) and 177Lu (PA-DOTA) -labelled IgG-CC-49 A long-term animal study was performed with 177Lu-labeled antibody utilizing its long half-life ( 161 hours). The experiment was performed on Balb / c mice for three weeks following the protocol set forth in Example XI. The results are shown in Table VIHA. See. pictures 29 - 34.

10 In the figures, where the results of the following tables are graphically represented, the following symbols are used:

Conjugates ΙΤ ^ Ζ | K “Vat 'Β1? 1' boli lock 'e.g.

15 i1; ^ Sn (EA-D03A)] - IgG-CC-iJ9 Δ IA 1-7 IX

(1:> -> SRj (EA-D03A)) - F (ab · 2) -CC-49 Δ 13 8-1 ») X

[?]; Sm (Ez-DTPA)] - IgG-CC-49? IC W ZA

['^ SmiBz-DTPA ^ -FUb ^ -CC ^ O ID 8-14 ZA

ί] H ^ Sm (PA-DOTA)] -IgG-CC-49 □ IIA 1-7 XII

[1: j-'Sm (PA-DOTA)) - F (ab'2) -CC-49 □ 113 6-14 XIII

20 l '^ Sm (BA-DOTA)] - I6G-CC-4 9 ΣΙΙΑ W XV

i1;> 3Sm (BA-DOTA)] - F (ab, 2) -CC-49 "JIIB 8-14 XVI

f) ·? ^ 3πΐ (PA-DOTMA)] -! gG - CC - 4? V VI IA 15-21 XXIV

[154Sm (PA-DOTMA)] - r (ab'2) -CC-49V VIIB 22-28 XXV

[1 '?' 7Lu (PA-D0TMA)] - IgG-CC-4O · VIIIA 29-34 XXIX

[rn Lu (PA-DOTA)] - IgG-CC-4 9V "viIIA 29-34 XXIX

: 25

The information shown in the figures below is as follows:.

119 104898

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.. en c · 2 <- tn o {v »· {M

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Claims (21)

A process for the preparation of a therapeutically active metal carbon black compound of formula (IA) R2 - // VS- (CH2) -C-M N'Q (IA) Q wherein each Q is independently hydrogen or (CHR5) pCO2R, 2. Process according to claim 1, characterized in that 1- [2- (4-isothioeyanatophenyl) ethyl] -1,4,7, -10-tetra-azacyclododecane-4 is used as a compound of formula IA , 7,10-triacetic acid. 3. A process according to claim 1, characterized in that 1- (2-methoxy-5-aminobentyl) -1,4,7,10-tetra-azacyclo- is used as a compound of formula IA; Of dodecane-4,7,10-triacetic acid. 3. Q1 and R2 are hydrogen and *. R 4 is nitro, or (H) the product of step (G) is reacted with a hydrogenation agent, such as Pd / C and hydrogen, under normal reaction conditions, to provide a compound of formula IA wherein R 3 is the same as defined above R 3 ', n is defined above, Q 1, R, R 2 and R 5 are hydrogen and R 4 is amino, or (I) a compound of the formula H yOMe / - \ Q1 \ / 10 // Wherein Q 1 is CO 2 R, R is C 1-6 alkyl and n is an integer from noli to five, is reacted with an α-halo carboxylic acid derivative of the formula BrCH 2 CO 2 R: wherein R is defined above, where an organic solvent and base are present at a temperature between 0 ° C and the reflux temperature, to provide a compound of formula IA wherein Q 1, Q and n are defined above. R 2 is hydrogen and R 4 is nitro, or (J) the product of step (I) is reacted with a deesterification agent, such as a strong acid, and then with a hydrogenation agent such as Pd / C and hydrogen. Reaction conditions to give a compound of formula IA wherein Q 1, Q and n are defined above, R, R 2 and R 5 are hydrogen and R 4 is amino, or (K) the product of step (B), ( F), (H) or (J) are reacted with the thiophosgene to provide the corresponding compound, wherein R 2 or R 4 is isothiocyanato; (L) the compound possibly of formula IA is reacted by known methods with a suitable reagent to effect potassium, sodium, lithium, ammonium, calcium, magnesium or hydrogen chloride additions of the compound as pharmaceutically acceptable salts, and ( M) a compound prepared according to any of the procedures (A) - (L) is reacted with a metal ion, which metal ion is La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho , Er, Tm, Yb, Lu, Y or Se. 3. R is C 1-4 alkyl and R is amino, or (C) the product of step (B) is reacted with a deesterification agent, such as a strong acid, to afford a compound of formula IA wherein 4. A process according to claim 1, characterized in that 1- (2-hydroxy-5-aminobentsyl) -1,4,7,10-tetra-azacy-clododecane-4 is used as a compound of formula IA. 7,10-triacetic acid. Process according to claim 1, characterized in that α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetra-azacyclododecane-1- (R, S-acetic) -4,7,10-tris- (R-methylacetic) acid 1-isopropyl-4,7,10-trimethyl ester. 104898 153 R 3, R 4 and R are hydrogen and R 2 is amino, or (G) a compound of the formula H (f 'N- (CH 2) -c-N N- h (IVA) n H / SO 2 Q 1 and n are as defined above, R 3, R 4 and R 5 are hydrogen, R is hydrogen and R 2 is amino, or (D) a compound of formula VA 10 defined above is reacted with an optically active α-benzenesulfonate of formula R * \ ^ C ° 0R 15 ▼ Where R 1a R 5 is defined above to provide a compound of formula IA wherein Q 1, R 5 and n are defined above, R 3 and R 4 are hydrogen, R is C 1. 4-alkyl and R 2 are nitro, or (E) the product of step (D) is reacted with a hydrogenation agent, such as Pd / C and hydrogen, under normal reaction conditions to provide a compound of the formula IA where Q 1, R 5 and n are defined above, R 3 and R 4 are hydrogen, R 3 is C 1-4 alkyl and R 2 is amino, or the product of step (E) is reacted with a deesterification agent , as a strong acid, to provide a compound of formula IA wherein Q1, R5 and n are as defined above, 6. Process according to claim 1, characterized in that α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetra-azacyclododecane-1 is used as a compound of formula IA - (R, S vinegar) -4,7,10-tris- (R-methylacetic) acid. 5 Process according to claim 1, characterized in that α- [2- (4-isothiocyanatophenyl) ethyl] -1,4,7,10-tetra-aza-cyclododecane-1- ( R, S-vinegar) -4,7,10-tris- (R-methylacetic) acid. 10 8. A process according to claim 1, characterized in that α- (4-aminophenyl) -1,4,7,10-tetra-azacyclododecane-1,4,7-triacetic acid is used as a compound of formula IA. 9. A process according to claim 1, characterized in that α- (4-aminophenyl) -1,4,7,10-tetra-azacyclododecane-1,4.10-triacetic acid is used as a compound of formula IA. 10. A process according to claim 1, characterized in that α- (4-aminophenyl) -1,4,7,10-tetra-azacyclododecane-1,4.7,10-tetraacetic acid is used as a compound of formula IA. 11. A process according to claim 1, characterized in that α- (4-isothiocyanatophenyl) -1,4,7,10-tetra-azacyclodo-1,4-decane-1,4 is used as a compound of formula IA , 7,10-tetraacetic acid. 12. Process according to claim 1, characterized in that α- [2- (4-nitrophenyl) ethyl] -1,4,7,10-tetra-azacyclododecane-1 is used as a compound of formula IA , 4,7,10-tetraacetic acid 1,4,7,10-tetramethyl ester. 30 13. A process according to claim 1, characterized in that α- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetra-azacyclododecane is used as a compound of formula IA -1,4,7,10-tetraacetic acid 1,4,7,10-tetramethyl ester. 14. A process according to claim 1, characterized in that o- [2- (4-aminophenyl) ethyl] -1,4,7,10-tetra-azacyclododone is used as a compound of formula IA. decane-1,4,7,10-tetraacetic acid. 15. A process according to claim 1, characterized in that α- [2- (4-isothiocyanatophenyl) ethyl] -1,4,7,10-tetra-azacyclododecane can be used as the compound of formula IA 1,4,7,10-tetraacetic acid. H is R 3 'is C 1-4 alkoxy, -OCH 2 CO 2 H or hydroxy and n is an integer from noil to five, reacted with an α-halo carboxylic acid of the formula BrCH 2 CO 2 H 2 where an organic solvent and base are present at temperature between 0 ° C and the reflux temperature to provide a compound of formula IA wherein R 3 is the same as defined above R 3 ', n is defined above; Q 1 is hydrogen or CO 2 R, each R is independently hydrogen or C 1-6 alkyl, provided that at least two of the substituents Q and Q 1 are other than hydrogen and that where Q 1 is hydrogen, R 3 is other than hydrogen, each R 5 is independently hydrogen or C 1-4 alkyl, n is an integer from noil to five, p is 1 or 2, R 2 is hydrogen or a nitro, amino, isothiocyanato, semi-carbazide, thiosemicarbazide, maleimide, bromoacetamide - or 1-carboxyl group, R 3 is C 1-4 alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen, R 4 is hydrogen or a nitro, amino, isothiocyanato, semi-carbazide, thiosemicarbazide, maleimide, bromoacetamide or provided that R2 and R4 cannot each be hydrogen but one of them must be hydrogen, or pharmaceutically acceptable salts thereof, complexed with a metal ion which is La, Ce, Pr, Nd, Pm, Sm, Eu , Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y tai Se, 35 characterized by having a compound having the accelerator H 5? ' Wherein Q 1 is CO 2 R, R is C 1-4 alkyl and n is an integer from no to ester, is reacted with an α-halo carboxylic acid ester of the formula Wherein R is -alkyl, where an organic solvent and base are present at a temperature between 0 ° C and the reflux temperature, to provide a compound of formula IA, wherein: Q1 and n are defined above; R 3, R 4 and R 5 are hydrogen, R is C 1-4 alkyl and R 2 is nitro, or (B) the product from step (A) is reacted with 3. a hydrogenation agent such as Pd / C and hydrogen under the reaction normal conditions, to provide a compound of formula IA wherein Q1 and n are defined above, R3, R4 and R5 are hydrogen, 16. A process according to claim 1, characterized in that o- (2-methoxy-5-nitrophenyl) -1,4,7,10-tetra-azacyclodeodecane-1 is used as a compound of formula IA , 4,7,10-tetraacetic acid. 17. Process according to claim 1, characterized in that α- (2-methoxy-5-aminophenyl) -1,4,7,10-tetra-azacyclo-dodecane-1 is used as a compound of formula IA. 4,7,10-tetraacetic acid in the form of tetra-ammonium acid. 18. Process according to claim 1, characterized in that o- (2-methoxy-5-isothiocyanatophenyl) -1,4,7,10-tetra-azacyclododecane-1,4 is used as a compound of formula IA. 7,10-tetraacetic acid in the form of tetraammonium salt. Method according to any of claims 1-18, characterized in that the metal ion is 153Sm, 166Ho, 90Y, 149Pm, 159Gd, 140La, 177Lu, 17SYb, 47Sc or 142Pr. 20. A method according to claim 19, characterized in that the metal ion is 153Sm, 177Lu or 90Y. 21. A process according to any one of claims 1 to 20, characterized in that the compound of formula IA is reacted with a metal ion by heating the reaction mixture. 30