NO179871B - Diagnostically usable macrocyclic complex compound - Google Patents

Description

Functionalized chelating agents, or bifunctional coordinating agents, are known to be capable of covalently binding to an antibody having specificity for cancer or tumor cell epitopes or antigens. Radionuclide complexes of such antibody/chelation conjugates can be used in diagnostic and/or therapeutic applications as a means of delivering the radionuclides to a cancer or tumor cell. See e.g. Meares et al., Anal. Biochem. 142. 68-78, (1984); and Krejcarek et al., Biochem. and Biophys. Res. Comm. 77, 581-585

(1977).

Aminocarboxylic acid chelating agents have been known and studied for many years. Typical of the aminocarboxylic acids are nitrile triacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), trans-1,2-diaminocyclohexanetetraacetic acid (CDTA) and 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA). Several bifunctional chelating agents based on amino-carboxylic acids have been proposed and prepared. E.g. is the cyclic dianhydride of DTPA [Hnatowich et al. Science 220. 613-615, (1983); U.S. patent 4,479,930] and mixed carboxy-carboxylic anhydrides of DTPA [Gansow, U.S. patents 4,454,106 and 4,472,509; Krejcarek et al., Biochem. and Biophys. Res. Comm. 77, 581-585, (1977)] have been described. When the anhydrides are connected to proteins, the connection takes place via the formation of an amide bond, leaving four of the original five carboxymethyl groups on the diethylenetriamine (DETA) backbone [Hnatowich et al. Int. J. Appl. Isot. 33, 327-332, (1982)]. Also, the U.S. describes patents 4,432,907 and 4,352,751-bifunctional chelating agents used to bind metal ions to "organic molecules such as organic target molecules or antibodies." As above, the coupling is achieved via an amide group using diaminotetraacetic acid dianhydrides. Examples of anhydrides include dianhydrides of EDTA, CDTA, propylenediaminetetraacetic acid and phenylene 1,2-diaminetetraacetic acid. A recent U.S. patent 4,647,447 describes several complex salts formed by the anion of a complexing acid for use in various diagnostic techniques. Conjugation via a carboxyl group in the complexing acid is described, giving a compound via an amide bond.

In J. of Radioanalytical chemistry 57 (12), 553-564 (1980), Paik et al. using p-nitrobenzyl bromide in a reaction with a "blocked" diethylenetriamine, i.e. bis-(2-phthalimidoethyl)amine followed by deblocking procedures and carboxymethylation using chloroacetic acid, to give N'-p-nitrobenzyldiethylenetriamine N,N,N" "N"-tetraacetic acid. Since the attachment is via a nitrogen atom, a tetra-acetic acid derivative is again obtained. Conjugation of the bifunctional chelating agent and chelation with indium are discussed. Substitution on the nitrogen atom is also described by Eckelman, et al.

in J. of Pharm. Pollock. 64(4), 704-706 (1975) by reacting amines such as "ethylenediamine or diethylenetriamine with the appropriate alkyl bromide prior to carboxymethylation." The compounds are proposed as potential radiopharmaceutical imaging agents.

Another group of bifunctional chelating agents based

on aminocarboxylic acid functionality is also well documented in the literature. Thus Sundberg, Meares, et al describe in J. of Med. Chem. 17(12), 1304 (1974) bifunctional analogues of EDTA. Representative of these compounds are 1-(p-amino-phenyl)-ethylenediaminetetraacetic acid and 1-(p-benzenediazonium)-ethylenediaminetetraacetic acid. Coupling to proteins via the para-substituent and binding of radioactive metal ions to the chelating group are discussed. The compounds are also described in Biochemical and Biophysical Research Communications 75(1) ,

149 (1977), and in U.S. patents 3,994,966 and 4,043,998. It is important to note that the attachment of the aromatic group to the EDTA structure is via a carbon atom in the ethylenediamine backbone. Optically active bifunctional chelating agents based on EDTA, HEDTA and DTPA are described in U.S. Pat. patent 4,622,420. In these compounds, there is an alkylene group that connects the aromatic group (which contains the necessary functionality for attachment to the protein) to the carbon atom in the polyamine that contains the chelating functionality. Other references to such compounds include Brechbiel et al. , Inorg. Chem. 25, 2772-2781 (1986), U.S. patent 4,647,447 and International Patent Publication No. WO 86/06384.

More recently, certain macrocyclic bifunctional chelating agents and the use of their copper chelate conjugates for diagnostic or therapeutic applications have been described in U.S. Pat. patent 4,678,667 and by Moi et al., Inorg. Chem. 26, 3458-3463 (1987). Attachment of the aminocarboxylic acid functionality to the rest of the bifunctional chelating molecule is via a ring carbon in the cyclic polyamine backbone. A connecting link, attached at one end to a ring carbon in the cyclic polyamine, is thus also attached at its other end to a functional group capable of reacting with the protein.

Another group of bifunctional chelating agents also worth noting consists of compounds in which the chelating group in the molecule, i.e. the aminocarboxylic acid, is linked via a nitrogen to the functional group in the molecule containing the group capable of reacting with the protein. As an example, Mikola et al describe in the patent application (International Publication Number WO 84/03698, published 9/27/1984) a bifunctional chelating agent prepared by reacting p-nitrobenzyl bromide with EDTA followed by reaction with bromoacetic acid to produce the aminocarboxylic acid. The nitro group is reduced to the corresponding amine group and then converted to the isothiocyanate group by reaction with thiophosphgene. These compounds are bifunctional chelating agents capable of chelating lanthanides that can be conjugated to bioorganic molecules for use as diagnostic agents. Since the attachment of the linker part of the molecule is via one of the nitrogen atoms of the aminocarboxylic acid, one potential aminocarboxylic group is lost for chelation. In this way, an EDTA-based bifunctional chelating agent containing four (not five) acid groups is produced. In this respect, this group of bifunctional chelating agents is similar to those where attachment to the protein is via an amide group with subsequent loss of a carboxyl chelating group.

Recently, Carney, Rogers and Johnson described (3rd. International Conference on Monoclonal Antibodies For Cancer; San Diego, California - 2/4-6/88) abstract entitled "Absence of Intrinsically 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 Density on the Biodistribution of Indium-111 Labeled B72.3 Immunoconjugates in Nude Mice". The biodistribution of indium-111 complexed with an EDTA and DTPA bifunctional chelating agent is described. The attachment of the aromatic ring to the EDTA/DTPA groups is via an acetate methylene. D. K. Johnson et al. [Florida Conf. on Chem. in Biotechnology, April 26-29 (1988), Palm Coast, FL] has also recently described in a meeting bifunctional derivatives of EDTA and DTPA where a p-isothiocyanate benzyl group is linked to the methylene carbon in one of the carboxymethyl groups. Previously, Hunt et al. in the U.S. patents 4,088,747 and 4,091,088 (1978) described ethylenediaminediacetic acid (EDDA) based chelating agents in which attachment of an aromatic ring to the EDDA group is via the alkylene or acetate methylene. The compounds are claimed to be able to be used as chelates to study hepatobiliary function. The preferred metal is technetium-99m. Indium-111 and indium-113m are also claimed to be able to be used as radionuclides for imaging.

It would therefore be advantageous to provide a complex that does not readily dissociate, is rapidly eliminated from the entire body except from the desired tissue, and conjugates with an antibody to produce the desired results.

In the accompanying drawings, the figures can be described as follows: Figures 1-7 and 15-21 show the biodistribution of 153Sm administered as a conjugate containing <153>Sm according to the present invention. The antibody CC4g-IgG was used in the conjugate according to the present invention. The biodistribution was determined in nude mice with LS 174-T tumor. Figures 8-14 and 22-28 show the biodistribution of 153Sm administered as a conjugate containing <153>Sm according to the present invention. The conjugate according to the present invention used CC4g-F(ab</>)2 as antibody fragment. The biodistribution was determined in nude mice with LS 174-T tumor. Figures 29-34 show the biodistribution of <177>Lu as a conjugate containing 17<7>Lu(PA-DOTMA) or 177Lu(PA-DOTA).

The conjugate used CC49-IgG as antibody. The biodistribution was determined in (balb/C) mice with LS 174-T tumor.

Surprisingly, the complexes and/or conjugates of the invention are relatively stable (ie they do not dissociate easily) and some show rapid clearance from the whole body and some non-target organs, such as liver, kidney and bone.

The invention includes the design and synthesis of new bifunctional chelating agents, all of which contain a chelating functionality, and a chemically reactive group for covalent binding to biomolecules. Part of the invention also constitutes methods for the preparation of various bifunctional coordinator (BFC) metal complexes and the binding of the complexes with antibody to produce antibodies labeled with radionuclides (such as samarium-153, lutetium-177 and yttrium-90) and/or fragments which suitable for diagnostic and/or therapeutic applications.

The present invention relates to novel bifunctional chelating agents which form complexes with metal ions, particularly "radioactive" metal ions having rare earth chemistry. Preferred rare earth metal ions include La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y and Sc, particularly preferred are Sm, Ho, Y and Sleep. Preferred rare earth radioactive metal ions include 153Sm, 166Ho, 90Y, 149Pm, 1<59>Gd, 140La, 17<7>Lu, <175>Yb, <47>Sc and 14<2>Pr, particularly preferred is 153Sm , <166>Ho, 9<0>Y and 17<7>Lu. Other radioactive metal ions that may be of interest are <4>7Sc, 99mTc, 186Re, 188Re, 97Ru, <105>Rh, 109pd> 197ptf 6<7>Cu> 198Au> 19<9>Au> 6<7>Ga, 68Ga, lllIn> 113<m>In,

<115>mIn, 117<m>Sn and <212>Pb/21<2>Bi. The complexes thus formed can be linked (covalently bound) to an antibody or fragment thereof, and used for therapeutic and/or diagnostic purposes. The complexes and/or conjugates can be formulated for in vivo or in vitro applications. A preferred application of the formulated conjugates is the treatment of cancer in animals, especially humans.

Applications of the complexes and/or conjugates according to this invention which contain a non-radioactive metal for diagnosis and/or treatment of disease states such as cancer are also possible. Such applications are known for non-radioactive metals using radio frequency to induce hyperthermia (Jpn. Kokai Tokkyo Koho JP 61,158,931) and fluorescent-immunoguided therapy (FIGS) [K. Pettersson et al., Clinical Chemistry 29(1), 60-64 (1983) and C. Meares et al., Acc.Chem. Res. 17, 202-209 (1984)].

More particularly, the present invention relates to a compound of the formula:

in which; each Q is independently hydrogen or (CHR<5#>)pCO 2 R; Q<1> is hydrogen or (CHR<5>')wCO 2 R; each R is independently hydrogen, benzyl or C1-C4 alkyl; provided that at least two of the sum of Q and Q<1> must be different from hydrogen; each R<5>' independently is hydrogen, C1-C4 alkyl or -(C1-C2 alkyl)phenyl; X and Y are both independently hydrogen or may be taken together with an adjacent X and Y to form an additional carbon-carbon bond; n is 0 or 1; m is an integer from 0 to 10; p = 1 or 2; r = 0 or 1; w = 0 or 1; provided that n is only 1 when X and/or Y form an additional carbon-carbon bond, and the sum of r and w is 0 or 1; L is a linker/spacer group covalently bound to, and replaces one hydrogen atom on one of the carbon atoms to which it is bound, said linker/spacer group being represented by the formula

in which:

s is an integer of 0 or 1;

t is an integer from 0 to 20;

R<1> is an electrophilic or nucleophilic group that allows covalent attachment to an antibody or fragment thereof, or a synthetic linker that can be attached to an antibody or fragment thereof, or precursor thereof; and

Cyc represents a cyclic aliphatic group, aromatic group, aliphatic heterocyclic group, or aromatic heterocyclic group, each of said groups being optionally substituted with one or more groups that do not interfere with binding to an antibody or antibody fragment;

provided that when s, t, i, r and n are 0, then R<1> is different from carboxyl; or

a pharmaceutically acceptable salt thereof.

Preferred features of the compounds of formula (I) are those where: R is hydrogen; R<5>' is H or methyl; n is 0; m is 0 through 5; r is 0; and L is a compound with the formula:

in which:

R<2> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido and maleimido;

R<3> is selected from the group consisting of C1-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;

R<4> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido and maleimide;

under the proviso that R<2> and R<4> cannot both be hydrogen, but one of R<2> and R<4> must be hydrogen; or

a pharmaceutically acceptable salt thereof.

When a conjugate of the present invention is desired, R<2> and R<4> must both be different from nitro. When R<2> or R<4> is nitro, then a precursor of the linker group (L) is present. This precursor group can be any group which is formed for R<2> or R<4> to prepare the compounds of formula (I) and which does not bind to an antibody or antibody fragment.

The present invention also relates to complexes of metal ions of the rare earth type, in particular radioactive neutral or charged complexes of metal ions of the rare earth type, and conjugates formed with the aforementioned complexes and antibody or antibody fragments. In addition, the present invention also includes formulations having conjugates according to the invention and a pharmaceutically acceptable carrier, especially formulations where the pharmaceutically acceptable carrier is a liquid. The invention also includes a method for the diagnosis or treatment of a disease state, especially cancer, in mammals which comprises administering to the mammal an effective amount of the formulation.

As used herein, the following terms shown have these meanings: with respect to the definition of R<1>, R<2> or R<4>, "electrophilic" groups include, but are not limited to, isothiocyanate, bromoacetamide, maleimide, imidoester , thiophthalimide, N-hydroxysuccinimyl ester, pyridyl disulfide and phenylazide; suitable "nucleophilic" groups include, but are not limited to, carboxyl, amino, acylhydrazide, semicarbazide, and thiosemicarbazide; "synthetic linkers" include any synthetic organic or inorganic linkers capable of being covalently attached to an antibody or antibody fragment, preferred synthetic linkers are biodegradable synthetic linkers which are stable in the patient's serum but have a potential for cleavage in a clearing agent for the radio-conjugate, e.g. biodegradable peptides or peptide-containing groups. Of the electrophilic groups, isothiocyanate is preferred, and of the nucleophilic groups, amino, semicarbazide and thiosemicarbazide are preferred. It is desirable that the character of and/or the position of R<1> is such that it does not significantly interfere with the chelation reaction.

The "X-C-Y" term in formula (I) represents the alternative presence of a double or triple bond between neighboring carbon atoms. The unsaturated bonds may be present independently in the chain length of 0 to 10 carbon atoms as defined by the "m" term in formula (I).

As used herein, the term "mammal" shall mean animals that feed their young with milk secreted by mammary glands, preferably warm-blooded mammals, more preferably humans. "Antibody" refers to any polyclonal, monoclonal, chimeric antibody or heteroantibody, preferably a monoclonal antibody; "antibody fragment" includes Fab fragments and F(ab')2 fragments, and any part of an antibody that has specificity for a desired epitope or epitopes. When the term "metal chelate/antibody conjugate" or "conjugate" is used, the "antibody" part shall mean that it includes whole antibodies and/or antibody fragments, including semi-synthetic or genetically engineered variants thereof.

As used herein, "complex" refers to a compound

according to the invention, e.g. formula (I), complexed with a rare earth metal ion, in particular a rare earth radioactive metal ion, in which at least one metal atom is chelated or complexed; "radioactive metal ion chelate/antibody conjugate" or "radioactive metal ion conjugate" refers to a radioactive metal ion conjugate covalently linked to an antibody or antibody fragment; "radioactive" when used in conjunction with the word "metal ion" refers to one or more isotopes of rare earth elements that emit particles and/or photons, such as 153Sm, 166Ho> 90y> 149pm> 159Gd> 140La> 177Lu> 175yb , 47Sc and 142pr;

the terms "bifunctional coordinator", "bifunctional chelating agent" and "functionalized chelating agent" are used interchangeably and refer to compounds having a chelating group capable of chelating a metal ion and a linker/spacer group covalently bound to the chelating group which is capable of serving as a means of covalently binding to an antibody or antibody fragment.

As used herein, "pharmaceutically acceptable salt" means any salt of a compound of formula (I) that is sufficiently non-toxic for use in mammalian therapy or diagnosis. The salts are thus usable in accordance with this invention. Representatives of these salts, which are formed by standard reactions, from both organic and inorganic sources, include e.g. sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, palmoic acid, mucinic acid, glutamic acid, d-camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, formic acid, lauric acid, stearic acid, salicylic- acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid and other suitable acids. Also included are salts formed by standard reactions of both organic and inorganic sources such as ammonium, alkali metal ions, alkaline earth metal ions and other similar ions. Particularly preferred are the salts of the compounds of formula (I) where the salt is potassium, sodium, ammonium or mixtures thereof.

The free acid of the compounds with the formula (I) can of course be used, also the protonated form of the compounds, e.g. when the carboxylate is protonated and/or the nitrogen atoms, i.e. when the HCl salt is formed.

Preferred compounds of the formula (I) include those compounds where Q<1> is hydrogen and L is represented by the formula A as shown by the following formula:

in which:

each Q is independently hydrogen or CHR<5>CO 2 R;

each R is independently hydrogen, benzyl or C1-C4 alkyl; provided that at least two of Q must be different from hydrogen;

m is an integer from 0 to 5;

R<2> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido and maleimide;

R<3> is selected from the group consisting of C1-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;

R<4> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido and maleimide;

each R<5> independently is hydrogen or C1-C4 alkyl;

under the proviso that R<2> and R<4> cannot both be hydrogen, but one of R<2> and R<4> must be hydrogen; or

a pharmaceutically acceptable salt thereof.

In formula (II) when R<3> and R<4> are both hydrogen, the compounds are represented by the following formula:

in which:

each Q is independently hydrogen or CHR<5>CO 2 R;

each R is independently hydrogen, benzyl or C1-C4 alkyl; provided that at least two of Q must be different from hydrogen;

m is an integer from 0 to 5;

R<2> is selected from the group consisting of nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido and maleimide;

each R<5 > independently is hydrogen or C 1 -C 4 alkyl; or

a pharmaceutically acceptable salt thereof.

In formula (II) when R<2> is hydrogen, the compounds are represented by the formula:

in which:

each Q is independently hydrogen or CHR<5>CO 2 R;

each R is independently hydrogen, benzyl or C 1 -C 4 alkyl; provided that at least two of Q must be different from hydrogen;

m is an integer from 0 to 5;

R<3> is selected from the group consisting of C1-C4-Alkoxy, - OCH2CO2H and hydroxy;

R<4> is selected from the group consisting of nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido and maleimide;

each R<5> independently is hydrogen or C1-C4 alkyl;

or

a pharmaceutically acceptable salt thereof.

Further preferred compounds of formula (I) include those compounds where at least one Q is hydrogen and are represented by the formula:

in which:

each Q is independently hydrogen or CHR<5>CO 2 R; Q<1> is hydrogen or (CHR<5>)wC02<R; >each R is independently hydrogen, benzyl or C 1 -C 4 -alkyl; under the condition that at least two of the sum of Q and Q<1> must be different from hydrogen and one of Q is hydrogen; m is an integer from 0 to 5; w is 0 or 1; R<2> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, maleimide and bromoacetamido; R<3> is selected from the group consisting of C1-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;

R<4> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl,

maleimide and bromoacetamido;

each R<5> independently is hydrogen or C1-C4-alkyl;

under the proviso that R<2> and R<4> cannot both be hydrogen, but one of R<2> and R<4> must be hydrogen; or

a pharmaceutically acceptable salt thereof.

Other preferred compounds of the formula (I) include compounds where Q<1> is CO 2 R (w=0) and are represented by the formula:

in which:

each Q is independently hydrogen or CHR<5>CO 2 R; each R is independently hydrogen, benzyl or C1-C4 alkyl; provided that at least one Q must be different from hydrogen; m is an integer from 0 to 5; R<2> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, maleimide and bromoacetamido; R<3> is selected from the group consisting of C1-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;

R<4> is selected from the group consisting of hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, maleimide and bromoacetamido;

each R<5> independently is hydrogen or C1-C4 alkyl;

under the proviso that R<2> and R<4> cannot both be hydrogen, but one of R<2> and R<4> must be hydrogen; or

a pharmaceutically acceptable salt thereof.

Some preferred compounds of formula (VI) are those where R<3> and R<4> are both hydrogen, the compounds are represented by the formula:

in which:

each Q independently is hydrogen or CHR<5>CC>2R;

each R is independently hydrogen, benzyl or C 1 -C 4 alkyl; provided that at least one Q must be different from hydrogen;

m is an integer from 0 to 5;

R<2> is selected from the group consisting of nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, bromoacetamido and maleimide;

each R<5> independently is hydrogen or C1-C4 alkyl;

or

a pharmaceutically acceptable salt thereof.

Other preferred compounds of the formula (VI) are those where R<2> is hydrogen and is represented by the formula:

in which:

each Q is independently hydrogen or CHR<5>CO 2 R; each R is independently hydrogen, benzyl or C 1 -C 4 alkyl; provided that at least one Q must be different from hydrogen; m is an integer from 0 to 5; R<3> is selected from the group consisting of C1-C4 alkoxy, -OCH2CO2H and hydroxy;

R<4> is selected from the group consisting of nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, carboxyl, maleimide and bromoacetamido;

each R<5> independently is hydrogen or C1-C4-alkyl;

or

a pharmaceutically acceptable salt thereof.

The bifunctional chelating agents described herein [represented by any of formulas (I)-(VIII)] can be used to chelate or complex rare earth metal ions, particularly radioactive rare earth metal ions, to form metal ion chelates ( also referred to herein as "complexes"). Because of. that the functionalization group (represented by "R<1>" in formula (I)] is present, the complexes can be attached to functionalized supports, such as functionalized polymeric supports or, when a complex of formula (I) where L represents formula (A) and R<2> and R<4> must be different from nitro, they may preferably be covalently attached to proteins or more particularly to antibodies or antibody fragments.The complexes described herein (represented by any of formulas I-VIII complexed with metal ions of the type rare earth species, especially radioactive metal ions of the rare earth type) can thus be covalently bound to an antibody or antibody fragment, and are referred to herein as "conjugates".

The antibodies or antibody fragments that can be used in the conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be prepared by hybridization techniques well known in the art, see e.g. Kohler and Milstein [Nature 256. 495-497 (1975); and Eur. J. Immunol. 6, 511-519 (1976)]. Such antibodies usually have a highly specific reactivity. Antibodies directed against any desired antigen or hapten can be used in the radioactive metal ion conjugates. Preferably, the antibodies used in the radioactive metal ion conjugates are monoclonal antibodies or fragments thereof which have high specificity for one or more desired epitopes. Antibodies used in the present invention can be directed against e.g. tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogenic surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules. Some examples of antibodies or anti-antibody fragments are CC-11, CC-46, CC-49, CC-49 F(ab')2, CC-83, CC-83 F(ab')2 and B72.3. [See D. Colcher et al., Cancer Res. 48. 4597-4603 (15 August 1988) for CC-49, CC-83 and B72.3 antibodies. The hybridoma cell line B72.3 is deposited in the American Type Culture Collection (ATCC) under accession number HB 8108. The various CC antibodies are described in U.S. Pat. patent application 7-073,685, filed July 15, 1987, which is available through NTIS. The other murine monoclonal antibodies bind to epitopes of TAG-72, a tumor-associated antigen]. A more complete list of antigens can be found in US patent 4,193,983, which is incorporated herein by reference. The radioactive metal ion conjugates according to the present invention are particularly preferred for the diagnosis and treatment of various types of cancer.

The preferred rare earth (lanthanide or pseudo-lanthanide) complexes according to the present invention are represented by the formula:

wherein: Ln is a rare earth (lanthanide) ion, such as Ce<3+>, Pr<3+>, Nd<3+>, Pm<3+>, Sm<3+>, Eu3+, Gd<3+ >, Tb<3+>, Dy<3+>, Ho3<+>, Er<3+>, Tm<3+>, Yb<3+> and Lu<3+>, or pseudo-lanthanide metal ion, such as

Sc<3+>, Y<3+> and La<3+>, particularly preferred metal ions are Y<3+>,

Ho<3+>, Lu<3+> or Sm<3+>; BFC represents a bifunctional chelating agent; and C represents a pharmaceutically acceptable ion or group of ions of sufficient charge to render the entire complex neutral. If the BFC contains four or more negatively charged groups, then C is a cation or a group of cations such as H+, Li<+>, Na<+>, K<*>, Rb<+>, Cs<+>, Mg< 2+>,

Ca<2+>, Sr<2+>, Ba<2+>, NH4<+>, N(CH3)4<+>» N(C2H5)4<+>, N(C3H7)4<+>, N(C4H9)4<+>, As(C6<H>5)4<+,> [(C6H5)3P=]2N<+> and other protonated amines. If the BFC contains three negatively charged groups, then C is not necessary. If BFC contains two negatively charged groups, then C is an anion such as F~, Cl~, Br", I", C104~, BF4~, H2P04~, HC03", HC02", CH3S03~, H3C-C6H4-S03" , PF6", CH3C02"" and B(C6H5)4~*

The conjugates of this invention, and in some cases the complexes of this invention, can be used as a formulation. The formulation comprises a compound of the formula (I) with the antibody and/or the metal ion and a physiologically acceptable carrier, a binder or aid for this. The formulation can thus consist of a physiologically acceptable carrier with a complex (metal ion + ligand), conjugate (metal ion + ligand + antibody) or (ligand + antibody). The methods for producing such formulations are well known. The formulation may be in the form of a slurry, injectable solution or other suitable formulations. Physiologically acceptable suspension media, with or without aids, can be used.

The formulations according to the present invention are in solid or liquid form containing the active radionuclide complexed with the ligand. These formulations can be in kit form so that the two components (i.e. ligand and metal,

complex and antibody, or ligand/antibody and metal) are mixed at an appropriate time prior to use. Either premixed or as a kit, the formulations usually require a pharmaceutically acceptable carrier.

Injectable compositions according to the present invention can be either in the form of a slurry or solution. When preparing suitable formulations, one must usually take into account that the water solubility of the salt is greater than the acid form. In solution form, the complex (or, if desired, the separate components) is dissolved in a physiologically acceptable carrier. Such carriers include a suitable solvent, preservatives such as benzyl alcohol, if necessary, and buffers. Applicable solvents include e.g. water, aqueous alcohols, glycols and phosphonate or carbonate esters. Such aqueous solutions do not contain more than 50% of the organic solvent by volume.

Injectable suspensions are compositions according to the present invention that require a liquid suspension medium, with or without auxiliaries, as a carrier. The suspension medium can be e.g. aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined mineral oils, or aqueous carboxymethylcellulose. Suitable physiologically acceptable auxiliaries, if they are necessary to keep the complex in suspension, can be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and the alginates. Many surfactants can also be used as suspending agents, e.g. lecithin, alkylphenol, polyethylene oxide adducts, naphthalene sulphonates, alkylbenzene sulphonates and the polyoxyethylene sorbitan esters.

Many substances that affect the hydrophilic properties,

the density, and the surface tension of the liquid suspension medium, may facilitate the production of injectable suspensions in some cases. E.g. silicone antifoams, sorbitol and sugars are all useful suspending agents.

An "effective amount" of the formulation is used for therapy. The dose will vary depending on the disease being treated. Although in vitro diagnosis can be carried out with the formulations according to this invention, in vivo diagnosis is also contemplated using formulations according to this invention. The conjugates and formulations according to this invention can also be used in radioimmunoguided surgery (RIGS); however, other metals that can be used for this purpose also include 99mTc, 11:LIn, <1>13mIn, <67>Ga and 68Ga.

Other uses of some of the chelating agents of the present invention may include magnetic resonance imaging, e.g. the binding to special purpose polymeric supports of complexes of formula I, especially complexes of formula VI, with Gd<+3>, e.g. as diagnostic agents, and removal of lanthanide metal or pseudo-lanthanide metal ion by selective extraction.

The present invention provides chelating agents, complexes and antibody conjugates some of which are more stable, and/or have improved biodistribution, and/or have faster clearance from the body than those known in the art. The methods for making the chelating agents, complexes and antibody conjugates of this invention are also provided.

A sensible and general synthetic approach to a twelve-membered macrocyclic, bifunctional chelating agent according to the present invention as represented by formula (I) entails the monofunctionalization of a free-base macrocycle (e.g. 1,4,7,10-tetraazacyclododecane) by only one of the nitrogen atoms with a suitable electrophile (eg any suitable substituted o-halocarboxylic acid ester). This electrophile must have a suitable linker group or suitable protected linker group that would allow covalent attachment of the bifunctional ligand to a protein, antibody or antibody fragment.

It is recognized in the art that alkylation techniques for the preparation of mono-N-functional polyaza macrocycles can result in mixtures of products that are difficult to separate [T. Kaden, Top.Curr.Chem. 121. 157-75 (1984)]. This problem has been solved by the described use of large excesses of macrocycle (5-10 equivalents in relation to the electrophile) which promotes formation of the monoalkylation adduct [M. Studer and T.A. Kaden, Helv.Chim.Act a 69. 2081-86 (1986); E. Kimura et al., J. Chem. Soc. Chem. Commun. 1158-59 (1986)].

Other routes to mono-N-functional polyazamacrocycles entail wide-ranging protection, functionalization and deprotection schemes [P.S. Pallavicini et al., J. Amer. Chem. Soc. 109, 5139-44 (1987); M.F. Tweedle et al., Eur. Pub. Pat. Appl. No. 0232-751]. A recent report on the reductive amination of substituted phenylpyruvic acids and amines has been published by Abbott Labs. as an abstract from a recent meeting (D.K. Johnson et al., Florida Conf. on Chem. in Biotechnology, April 26-29 (1988), Palm Coast, FL). A process that prepares mono-N-alkylated polyaza macrocycles using an electrophile with between about one and five equivalents of a suitable macrocycle in a solvent that will not promote proton transfer is described in U.S. Pat. Application Serial No. 289,163, by W.J. Kruper, filed Dec. 22, 1988.

General synthetic routes to the chelating agents according to the present invention are described in synthesis scheme I-IV

in the following, and involves reaction of a suitable electrophile and the polyazamacrocycle, at different stoichiometry, temperature and concentration in a suitable organic solvent. Examples of suitable organic solvents are any hydrocarbon that supports solubility, such as acetonitrile, isopropanol, methylene chloride, toluene, chloroform, n-butanol, carbon tetrachloride, tetrahydrofuran, 5% ethanol in chloroform, while the most preferred are chloroform, methylene chloride, n -butanol, 1,4-dioxane and acetonitrile. Stoichiometric amounts or near-stoichiometric amounts of macrocycle and electrophile can be used, giving the corresponding mono-N-alkylation product in a single step. The temperature range for the reaction is from about 0°C to about reflux, preferably from 0°C to 25°C. The reaction time to complete turnover is about 1 to about 24 hours.

General access to substituted α-haloacid esters is desirable in order for synthesis schemes II and IV to be usable overall. A suitable approach entails bromination or chlorination of the in situ produced acid halide, e.g.

D. N. Harpp et al., J. Org. Chem. 40, 3420-27 (1975). This method allows exclusive alpha-halogenation of alkanoic acids even containing reactive benzyl groups. A general method for substituted acid halides involves reacting the organic acid with thionyl chloride or sulphuryl chloride, e.g. E. Schwenk et al. J. Amer. Chem. Soc. 70, 3626-27

(1944). Both methods use the free carboxylic acid which is often available from commercial sources.

Polyazamacrocycles such as 1,4,7,10-tetraazacyclododecane can be prepared by documented methods such as T. J. Atkins et al., J. Amer. Chem. Soc. 96, 2268-70 (1974) and T.J. Richman et al., Org. Synthesis 58, 86-98 (1978).

Carboxymethylation of the mono-N-functional macrocycle can be carried out by the method of Desreux using bromoacetic acid derivatives and a suitable base [J.F. Desreux, Inor. Chem. 19. 1319-24 (1980)].

All the starting materials which are necessary for the production of the compounds according to this invention are either available from commercial sources or can be made from descriptions in known literature references.

In the following scheme (I), the compounds are prepared with the formula (I) where Q<1> is hydrogen. Although only one compound is indicated by the conditions shown, other similar groups can also be prepared within formula (I) where Q<1> is hydrogen, r = 0 or 1, n = 0 or 1 and m = 0 through 10 using this method.

In the following form II, the compounds with the formula (I) are prepared where Q<1> is hydrogen. Although only one compound is indicated by the conditions shown, other similar groups can also be prepared within formula (I) where Q<1> is hydrogen, r = 0 or 1, n = 0 or 1 and m = 0 through 10 using this method.

In the following form III, the compounds with the formula (I) are prepared where Q<1> is CO 2 R. Although only one compound is shown under the stated conditions, other similar moieties can also be prepared within formula (I) wherein Q<1> is CO 2 R, including m = 0 through 10, n = 0 or 1 and r = 0 using this procedure.

In the following form IV, the compounds of the formula (I) are prepared where Q<1> is (CHR<5>')wC02R. Although only one compound is shown under the stated conditions, other similar groups can also be prepared within formula (I) where Q<1> is (CHR<5>')wC02R, comprising m = 0 through 10, n = 0 or 1 and r = 0 by this procedure.

In Scheme IV, the reagent shown containing A' can be any suitable leaving group for a cx-acid, ie A' is phenyl or CF 3 .

Use of an optically active alkylating agent has minimized the diastereomers and simplified the synthesis. Isolation of a single diastereomer that could lead to a single lanthanide complex that was easy to purify would be desirable for radical chelation as well as for antibody conjugation.

In the following scheme V, the compounds are prepared with the formula (I) where Q<1> is hydrogen or (CHR<5>)wC02<R> and R<2> is hydrogen. Although only two compounds are shown at the stated conditions, other similar groups within formula (I) where Q<1> is hydrogen or (CHR<5>)wC02R, Q is hydrogen or (CHR<5>)pC02R, r = 0 or 1, n = 0 or 1, w = 0 or 1, m = 0 through 10, p = 1 or 2, L represents the formula A wherein R<3> is defined as before, R<2> and R<4> is selected from the group consisting of hydrogen, amino, nitro, isothiocyanate, semicarbazide, thiosemicarbazide, maleimide, bromoacetamido and carboxyl, is prepared by this method.

The electrophilic group ("R<1>" in the formula) can also be prepared by methods known in the art. Such procedures can be found in Acc. Chem. Res. 17» 202-209 (1984). A method which can prepare compounds of the formula

(I) where R<1> could be an isothiocyanate group (present R<2> or R<4> is isothiocyano when L = formula A) leads to reaction of the amino chelate (R<2> or R<4> equal to amino, present when L =

formula A) with thiophosgene, and is described in U.S. Pat. application series no. 289,172, by M.J. Fazio, et al., filed December 23, 1988.

Radionuclides can be produced in several ways. In a nuclear reactor, a nuclide is bombarded with neutrons to achieve

a radionuclide, e.g.

Sm-152 + neutron Sm-153 + gamma

Another method of obtaining radionuclides is to bombard nuclides with particles in a linear accelerator or a cyclotron. Yet another way is to isolate the radionuclide from a mixture of fission products. The method of obtaining the nuclides used in the present invention is not decisive for them.

The conjugates according to the present invention can be prepared by first forming the complex and then binding the antibody or antibody fragment. The method thus involves preparing or obtaining the ligand, forming the complex with the metal and then adding the antibody. Alternatively, a method for preparing labeled antibody conjugates may first involve conjugation of BFC to the antibody and its subsequent chelation to obtain the radionuclide BFC-labeled Ab. Any suitable method leading to the formation of the conjugates of this invention is within the scope of this invention.

In the following examples, the following terms and conditions were used, if nothing else is stated.

Mass spectra were obtained on either a Finnigan TSQ mass spectrometer (Q^-MS method) or a high-resolution VG ZAB-MS mass spectrometer (fast atom bombardment with xenon, using 3:1 dithiothreitol:dithioerythritol).

^-H and <13>C NMR spectra were obtained using a Varian VXR-300, Bruker APC 300, IBM/Bruker NR-80 or a Jeol FX400 spectrometer. All spectra were prepared at 30°C unless otherwise noted. <X>H NMR was performed at resp. 300 MHz, 80 MHz or 400 MHz, on the equipment listed above; <13>C NMR was performed at resp. 75 MHz, 20 MHz or 100 MHz on the equipment listed above. The values for NMR are S versus TMS (tetramethylsilane) or when D 2 O was the solvent, versus DSS (2,2-dimethyl-2-silapentane-5-sulfonic acid, sodium salt).

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

For the chromatography procedures, most solvents were Fisher HPLC grade materials. Ammonium acetate was purchased from Aldrich. Water was purified using a Barnstead NANOpure™ water filtration system. Preparative chromatography of organic compounds was performed either by conventional gravity chromatography using standard techniques, or by flash chromatography as described by C.W. Still et al., J. Org. Chem.

43., 2923-24 (1978). The following solvent systems were used:

Rf values are reported using these solvent systems and commercially available normal phase silica TLC plates [GHLF 250 micron, Analtech Inc. or Merck Kieselgel 6OF254]. Preparative column chromatography was performed using Merck grade 60, 60 Å silica gel.

All percentages are by weight unless otherwise noted.

Some solids were dried using a rotary evaporator (Buchi 461) and/or a vacuum oven at a temperature of about 55-60°C for several hours. Additionally, a Virtis model 10-010 automatic freeze dryer or Speed Vac™ concentrator was used for solvent removal.

Samarium-153 and lutetium-177 were produced by the research reactor, University of Missouri, (Columbia, MO). Yttrium-90 was purchased from Oak Ridge National Laboratory.

1-(4-isothiocyanatebenzyl)diethylenetriaminepentaacetic acid (SCN-Bz-DTPA) was prepared by a modification of the method of M.W. Brechbiel, et al., Inorg. Chem. 25, 2772-2781

(1986) , and purified to give a single compound by anion-exchange HPLC (Q-Sepharose™). Some of the chemicals used were supplied from the indicated sources: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), free acid and the sodium salt were 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.

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 GF-250 (9.4 mm x 25 cm) from Du Pont Instruments; Vydac™ (Trademark of the Separations Group, Hesperia, CA) protein C-4 (4.6 mm x 25 cm) from the Separations Group (Hesperia, CA); and Mono-Q™ and SP-Sephadex™ (Trademark of Pharmacia Biotechnology Products) from Pharmacia. Sep-Pak™ (trademark of Waters Associates) C-18 cartridge was purchased from Waters Associates (Milford, MA), Sephadex™ G-25 disposable columns

(2.2 mL) from Isolab Inc. (Akron, OH), and Centricon™-30

(Trademark of Amicon Division, W. R. Grace & Co., Danvers, MA)

micro concentrators from Amicon.

Five HPLC systems were used for analysis and sample separations: System I consisted of LKB 2150 pump, and 2152 controls, a UV detector - LKB 2238 UV Cord, a Berthold LB 506 A HPLC radioactivity monitor (from the international Berhold group)

and a Gilson fraction collector 201-202 (Gilson International, Middleton, WI);

System II was equipped with an auto-sampler, the WISP 710 B,

two pumps (model 510) and an automatic gradient controller (from Waters Associates), a Perkin-Elmer LC-75 spectrophotometric detector, a Beckman model 170 radioisotope detector, and a fraction collector (Frac-100 from Pharmacia);

System III consisted of two Waters M-6000A pumps with a Waters 660 solvent programmer, a Waters model 481 variable wavelength detector, and a Waters data module, also an ISCO fraction collector was used preparatively.

System IV was the Dionex BioLC™ system equipped with a variable wavelength UV detector; and

System V was a Waters model 590 pump with an ISCO model 2360 gradient programmer and a Dionex variable wavelength detector.

A Sorvall RT 6000B (refrigerated centrifuge from Du Pont) was used for centrifugation and cocentrifugation. A Speed Vac concentrator (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.) CRC-12, or by liquid scintillation on a Beckman LS 9800 (Beckman Instruments, Inc, Irvine, CA), or on a Canberra 35<+> multichannel analyzer phased with a 3 inch (7.5 cm) thallium powered sodium iodide well crystal.

In the examples concerning complex formation, the percent complex determination was carried out using the following general exchange method where indicated.

A 10 ml plastic column was filled with 1 to 2 ml SP-Sephadex™ C-25 resin swollen in water. Excess water was removed by pressure at the top of the column. The test solution

(15 µl) was added to the top of the resin, then 2 mL of 4:1 (V:V) isotonic saline was used: conc. NH4OH as eluent. The eluents were collected in a counting tube. An additional 2 ml of eluent was used. After collecting the eluent in a second counting tube, air pressure was applied to the top of the resin to bring it to dryness. The dried resin was transferred to a third counting tube. The activity in each of the counting tubes was measured with a Nal well counter connected to a Canberra multichannel analyser. The amount of the metal complexed was given as a percentage of the total activity found in the eluent. The amount of metal in the free uncomplexed state was given as the percentage of all the metal remaining in the resin.

Removal of uncomplexed metal by ion exchange<g>

A 10 ml plastic column was filled with 0.5 ml SP-Sephadex™ C-25 resin swollen in water. Excess water was removed by centrifugation of the column at 3,000 rpm for 4 min. A volume of 200-500 µl of the complex solution was placed on top of the resin, and the tube centrifuged again for 4 min. at 3,000 rpm. The uncomplexed metal was left in the column and the complexed metal was eluted with the solvent.

Preparation of yttrium complex:

Complexes were made by preparing a 3 x 10~<4>M yttrium solution in water (YC13.6H20, 303.26 g/mol; or Y(0Ac)3, 12.1% H20). Radioactive Y<3+->solution (Oakridge National Laboratories) was added to give the desired level of radioactivity. 10 µl of ligand solution (at 0.03M) was added to 990 µl of the Y<3+ -> solution, giving a 1:1 molar ratio of ligand:metal. 10 times the amount of ligand solution was used for a 10:1 ligand to metal ratio. The pH was adjusted to 7.4 using microliter amounts of HCl or NaOH. The solution was then tested for the amount of complexed yttrium using the cation exchange method given above.

Preparation of samarium complex:

Samarium complexes were made as described above for yttrium complexes, except that 3 x 10~<4>M samarium was prepared by dissolving Sm 2 O 3 (348.7 g/mol) in 0.1 M HCl. Radioactive Sm-153 was prepared as an approximately 3 x 10<4>M solution in 0.1M HCl from the University of Missouri Research Reactor, Columbia, Missouri.

The following definitions are given for some of the expressions used in this text.

Dictionary:

conc. means concentrated;

~OAc means the acetate group, "0C0CH3;

TLC means thin layer chromatography;

DI H20 means deionized NANOpure™ water;

NANOpure™ water is pure water obtained from a Barnstead NONOpure™ water filtration system;

Ambient temperature means room temperature or about 20 to about 25°C;

Overnight means from about 9 to about 18 hours;

LC means liquid chromatography;

NBS means N-bromosuccinimide;

MES means 2-(N-morpholine)ethanesulfonic acid;

HPLC means high-pressure liquid chromatography;

PBS means phosphate buffered saline from Sigma, containing 120 mM NaCl, 2.7 mM KCl and 10 mM phosphate buffer, pH 7.4;

SP-Sephadex™ C-25 resin is a cation exchange resin having sulfonic acid functionality and is sold by Pharmacia, Inc.;

rpm = revolutions per my.;

pD means the pH at which the hydrogen is deuterated;

DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

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

BFC = bifunctional chelating agent;

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 = oc-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic) acid;

BA-DOTA = α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

OMe-BA-DOTA = c -(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-isothiocyanatebenzyl)-diethylenetriaminepentaacetic acid;

EDTA = ethylenediaminetetraacetic acid;

chelating agent is equivalent to ligand;

complex is equivalent to chelate;

conjugate means a chelating agent or complex covalently linked to an antibody or antibody fragment; and

antibodies means CC-49, CC-83 and B72.3 and their fragments such as Fab and F(ab')2. Other possible antibodies have been provided herein previously. The hybridoma cell line B72.3 is deposited in the American Type Culture Collection, with the accession number ATCC HB 8108, and the other named murine monoclonal antibodies bind to epitopes of TAG-72, a tumor-associated antigen.

The invention will now be further elucidated by considering the following examples, which are solely intended to be examples of the application of the invention.

Production of starting materials

Example A

Preparation of d,l-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 brought to reflux for 1 hour with the beginning of evolution of hydrogen chloride gas and sulfur dioxide gas. To the hot solution was added 11.0 g (0.06 mol) of N-bromosuccinimide in 25 ml of carbon tetrachloride, and three drops of 48 percent aqueous hydrogen bromide catalyst. Release of bromine gas was noted. The dark red solution was brought to reflux for 35 min. The solution was cooled and poured into 100 ml of methanol with stirring. TLC analysis (60:40 ethyl acetate:hexane v:v) indicated a new product (Rf = 0.69, silica plates). Excess solvent was removed by rotary evaporation and the dark red oil was filtered through a flash silica gel pad (1 inch x 5 inch).

(2.5 cm x 12.5 cm) using methylene chloride as eluent. Evaporation of the solvent gave a clear oil, yield 15.43 g, which was an 85:15 mixture of the title product: the methyl ester of the original butanoic acid derivative. The title product was characterized by:

<x>li NMR (CDC13)

8.16(d), 7.38(d), 4.20(dd), 3.79(s), 2.88(m), 2.38(m);

<13>C NMR (CDCl3, 75 MHZ)

169.6, 147.5, 129.3, 123.7, 53.0, 44.4, 35.5, 33.0.

Example B

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

2.07 g (5.82 mmol) of d,l-2- bromo-4-(4-nitrophenyl)butanoic acid, methyl ester (prepared by the procedure in Example A) during five minutes under a nitrogen atmosphere with stirring. The reaction mixture was stirred for 48 hours at about 25°C. TLC (using solvent system 2, on Analtech silica gel plates) indicated formation of the title product (Rf = 0.73). The yellow chloroform solution was applied to a 1 inch x 15 inch flash silica gel column (pre-eluted with 5% methanolic chloroform), eluted with 250 mL of 5% methanolic chloroform, followed by elution with solution medium 2. Fractions containing pure title product were pooled, and evaporated to give 2.15 g (5.46 mmol, 94%) of the title product as a pale yellow glass. Rubbing a chloroform solution of this glass in diethyl ether resulted in the precipitation of a white powder (m.p. = 156-159°C) of analytical purity and characterized by:

<1->H NMR (CDCl3)

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 (CDC13 > 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)ethanoic 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 at reflux under a N 2 atmosphere for three hours. The mixture was then evaporated to dryness in vacuo. The residue was dissolved in carbon tetrachloride and stirred at reflux under a nitrogen atmosphere. Bromine (7.2 mL, 0.14 mol) was added in small portions over a period of 3 days to the reflux mixture. The reaction mixture was allowed to cool and the acid chloride was quenched with methanol (50 mL, added slowly).

The resulting bridge master (27 g, 71%) was recovered as a

yellow oil by distillation at reduced pressure (bp = 168°C at 1.1 mm) through a 6 inch column of glass spirals. The structure of the title product was confirmed by:

3-H NMR (CDCl3)

8.25(d), 7.81(d), 5.51(s), 3.86(s).

Example D

Preparation of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1-acetic acid, 1-methyl ester.

To 529 mg (2.068 mmol) of 2-bromo-2-(4-nitrophenyl)ethanoic acid, methyl ester (prepared by the procedure in Example C) in 20 ml of acetonitrile was added at once a solution of 354 mg (2.068 mmol) of 1 ,4,7,10-tetraazacyclododecane in 20 ml of acetonitrile which contained just enough methanol to effect complete dissolution. The resulting pale pink solution was stirred for 1.5 hours, then concentrated in vacuo at 35°C to a small volume. This crude monoester tetraamine was purified by silica gel chromatography, eluting with 20% (w/v) NH 4 OAC/CH 3 OH. The purified monoester tetraamine isolated in this way as the triacetate salt was then converted to the free amine. Then 270 mg in 3 mL of water was treated at 0°C with aqueous K 2 CO 3 (10% w/w) to a pH of 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 concentrated to give 160 mg (42% yield) of the desired monoester tetraamine, characterized by NMR and fast atomic bombardment mass spectrometry ([M+H]<+>=366) and by:

<X>U NMR (CDCl3)

8.12(d), 7.44(d), 4.75(8), 3.76(s), 2.67-2.44(m);

<13>C NMR (CDCl3)

171.5, 147.6, 144.0, 130.,0, 124.0, 67.0, 49.8, 47.9, 46.9, 46.0.

Example E

Preparation of N-(2-methoxy-5-nitrobenzyl)-1,4,7,lo-tet raw 2 acyclic ododecane.

To a stirred chloroform solution (20 ml) containing 2.9 g of 1,4,7,10-tetraazacyclododecane (16.8 mmol) was added a chloroform solution (20 ml) containing 2.1 g of 2-methoxy-5-nitrobenzyl bromide (8.5 mmol) in one portion. After stirring at room temperature for 3 hours, the reaction mixture was filtered, and . the filtrate concentrated (in vacuo) to give a residue which was chromatographed (silica, solvent system 3). The mono-alkylated product was isolated in 79% yield (m.p. = 154-156°C), and characterized by:

<13>C NMR (CDCl3)

162.47, 140.63, 127.92, 125.49, 124.53, 109.91, 55.88, 53.25, 50.90, 47.18, 45.45, 45.29.

Example F

Preparation of N-(2-hydroxy-5-nitrobenzyl)-1,4,7,lo-tet raazacyclododecane.

To a 1,4-dioxane solution (20 mL) of 1,4,7,10-tetraazacyclododecane (2.3 g, 14 mmol) was added a dioxane solution (15 mL) of 2-hydroxy-5-nitrobenzyl bromide (1,2 g, 7 mmol) in one portion with constant stirring. After several minutes, K 2 CO 3 (lg) was added, and stirring continued for 1 hour at room temperature. The reaction mixture was then filtered, and the filtrate concentrated in vacuo to give a yellow semi-solid which was chromatography (column) on silica gel eluted with solvent system 5. The desired mono-alkylated product was isolated from the last 1/3 of pale yellow eluent which also contained unreacted amine. After concentration, the yellow oil was triturated with CHCl 3 (100 mL) and filtered to remove CHCl 3 -soluble amine starting material. The final product was then isolated in pure form as a yellow solid (1.2 g, 55%); (sp. = 142-145°C) and further characterized by:

<13>C NMR (D2O)

25.04, 25.28, 26.83, 31.80, 36.54, 101.88, 107.22, 110.92, 111.80, 115.26, 159.99.

Example G

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

To a stirred solution of 3.50 g (20.3 mmol) of 1,4,7,lo-tet raa zacycl ododecane in 50 ml of pentene-stabilized chloroform was added dropwise over a 5 minute period with vigorous stirring under a nitrogen atmosphere 4 .00 g (17.4 mmol) of 1-(2-bromomethyl)-4-nitrobenzene.

Stirring was continued for about 18 hours at room temperature (about 25°C), whereupon crystals of amine hydrobromide fell out of the solution. The entire reaction mixture was applied to a flash silica gel column (1 inch x 18 inch) that had been pre-eluted with 5% methanol in chloroform; 200 ml of this solution was added as eluent, followed by elution with solvent system 3. 1.45 g (9.7 mmol) of p-nitrostyrene (Rf = 0.98; solvent system 1) was separated from the desired product ). The desired monofunctional title product was isolated as an orange-yellow oil (2.17 g, 7.06 mmol, 40.6%) which solidified on standing (Rf = 0.73, solvent system 1).

A sample of the title product was recrystallized from CHCl3/cyclohexane and showed the following characteristics: m.p. = 146.5-148.5°C;

<X>H NMR (CDCl 3 )

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

2.72(h), 2.50(h), 2.60(s);

<13>C NMR (CDCl3, 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-tetraazacyclododecane.

Into 50 ml of chloroform were added 3.5 g (20.3 mmol) of 1,4,7,10-tetraazacyclododecane and 1.7 g (7.87 mmol) of p-nitrobenzyl bromide, and the mixture was stirred under nitrogen for 24 hours at 25°C. The chloroform slurry of the hydrobromide salt was then applied to a 1 inch x 17 inch column of flash silica gel (solvent system 3). 2.13 g (6.93 mmol) were obtained

of the title product as a pale yellow solid of analytical purity in 88% yield (Rf = 0.58, solvent system 3), m.p. = 128-129°C, and further characterized by:

3-H NMR (CDCl3)

8.19 (d), 7.49 (d), 3.69 (s), 2.82 (t), 2.70 (t), 2.59 (m);

<13>C NMR (CDCl3, 75 MHz)

147.2, 128.4, 123.8, 58.8, 51.7, 47.1, 46.3, 45.1.

(No example I; to avoid confusion with later biology example numbers).

Example J

Preparation of 1-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane.

690 mg (2.25 mmol) of 1-(4-nitrobenzyl)-1,4,7,10-tetraazacyclododecane (prepared by the method in example H) were dissolved in 10 ml of methanol. 350 mg of 10% palladium on carbon catalyst was added to the solution. Excess hydrogen was bubbled through the solution at 25°C. Within 45 min. TLC showed that the title product was produced (Rf = 0.33, solvent system 3). Chromatographic purification of the title product formed was carried out on a 1 inch x 16 inch flash silica column (solvent system 3) to give 480 mg (77%) of the title product as a pale yellow clear oil.

The free base title product (103 mg, 0.37 mmol) was converted to the hydrochloride salt by bubbling anhydrous hydrogen chloride through ethanol solution of the free base. The resulting tetrahydrochloride salt of the title product, after washing with cold ethanol and drying in vacuo, was obtained in a yield of 147 mg (0.347 mmol), m.p. = 255-260°C (dec), and was further characterized by:

^-H (D2O, pD = 1.9)

7.56 (d), 7.47 (d), 3.95 (s), 3.28 (t), 3.23 (t), 3.09 (t), 2.96 (t);

<13>C NMR (D2O, pD = 1.9, 75 MHz)

138.3, 134.2, 132.2, 126.0, 58.5, 50.3, 46.7, 44.6, 44.3.

Example K

Preparation of 2-methoxy-5-nitrobenzylnitrile.

To a solution of sodium cyanide (6 g, 121.9 mmol) in water

(5.3 ml) at 70°C was added in small portions with stirring a hot solution of 2-methoxy-5-nitrobenzyl bromide (25 g, 101.6 mmol) in ethanol (15 ml). The reaction mixture was then refluxed for 1.5 hours, cooled and filtered. The filter cake was washed with acetonitrile and the filtrate was evaporated in vacuo

to give 2-methoxy-5-nitrobenzylnitrile (19.5 g, 100%) as a tan solid. The product was characterized by:

<13>C NMR (CDCl3)

161.4, 141.0, 125.7, 124.6, 119.8, 116.5, 110.2, 56.3, 18.8.

Example L

Preparation of 2-methoxy-5-nitrophenylacetic acid.

A slurry of 2-methoxy-5-nitrobenzylnitrile (19.5 g)

(prepared by the method in example K) in conc. HCl (20 mL) was refluxed for 3 h. After cooling, the crude product was recovered by filtration and washed with water. The solid was taken up in hot aqueous sodium hydroxide and filtered hot. The orange solution was cooled and acidified with HCl. The precipitate was filtered, washed with water, and dried to give 2-methoxy-5-nitrophenylacetic acid as a white solid (15 g, 70%). The product was characterized by:

<13>C NMR (CDCl3-CD3OD)

172.8, 162.5, 140.7, 126.2, 124.7, 124.1, 109.7, 55.8, 35.0.

Example M

Preparation of α-bromo-(2-methoxy-5-nitrophenyl)acetic acid, methyl ester.

To a slurry of 2-methoxy-5-nitrophenylacetic acid (2.266 g, 10.74 mmol) (prepared by the procedure in Example L) in dry benzene (50 mL) was added thionyl chloride (4.0 mL, 54.8 mmol).

A drying tube and condenser were placed on the bottle and the mixture was refluxed for 2 hours. The resulting yellow solution was concentrated in vacuo to a small volume and taken up in dry carbon tetrachloride. Bromine (0.6 mL, 11.6 mmol) was added and the solution was refluxed for two days while excluding atmospheric moisture. The reaction mixture was concentrated to a small volume and methanol (50 mL) was added. After evaporation of excess methanol in vacuo, the resulting oil was purified chromatographically (silica gel, methylene chloride-hexane 4:1). The title product (2.399 g, 74%) was obtained as a yellow oil. The product was characterized by:

1 H NMR (CDCl3)

8.45(dd), 8.15(dd), 6.95(d), 5.75(s), 3.99(s), 3.78(s).

Example N

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

To a stirred solution of 1.4 ,7,10-tetraazacyclododecane (2.72 g, 15.79 mmol) in chloroform (50 mL). The mixture was stirred at room temperature for 2 hours.

The cloudy mixture was then cocentrated to a small volume in vacuo at room temperature. The crude product was purified by chromatography (silica gel, solvent system 3). The title compound was recovered as a yellow solid (2.60 g,

83%) and characterized by:

<13>C NMR (CDCl3)

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 O

Preparation of d,1-2-bromo-4-(4-nitrophenyl)-butanoic acid iso-propyl ester.

4-(4-Nitrophenyl)butanoic acid (21.0 g, 0.10 mol) was added to a solution of carbon tetrachloride (10 mL) and thionyl chloride (30 mL, 0.4 mol) under a nitrogen atmosphere using the method of Harpp et al., J. Org. Chem 40, 3420-27 (1975). The solution was brought to reflux for 1 hour with the initial rapid release of hydrogen chloride and sulfur dioxide. At this point, N-bromosuccinimide (22.0 g, 0.12 mol) was added as a solution in carbon tetrachloride (50 mL) and 8 drops of 48% aqueous hydrogen bromide catalyst were added to the hot solution, whereupon liberation of bromine was noted. The dark red solution was refluxed for a further 35 min. The solution was cooled and poured into isopropanol (400 ml) with stirring. TLC analysis (methylene chloride, silica gel plates)

revealed a new product (Rf=0.73). Excess solvent was removed and the dark red oil was filtered through a flash silica gel pad (3 inches x 6 inches) using methylene chloride as eluent. The solvent was removed in vacuo and the pale yellow oil was applied to a flash silica gel column (3 in. x 18 in.) and eluted with methylene chloride to give 25.0 g (0.076 mol) of the title bromester product as a clear oil in 75% yield. as an isopropanol solvate containing less than 5% unbrominated ester and characterized by:

<X>H NMR (CDCl 3 )

8.16(d, 2H), 7.38(d, 2H), 5.05(septet, 1H), 4.14(dd, 1H), 2.88(m, 4H), 2.39(m , 4H), 1.29 (d, 6H);

<13>C NMR (CDCl3)

168.7, 147.7, 129.3, 123.8, 69.9, 45.1, 35.6, 33.0, 21.5, 21.2.

Example P

Preparation of c-[3-(4-nitrophenyl)propyl]-1,4,7,10-tetraazacyclododecane-1-acetic acid, 1-methyl ester.

To a stirred solution of 3.137 g (18.2 mmol) of the cycle free base in 30 ml of pentene-stabilized chloroform was added 4.81 g (14.6 mmol corrected) of d,l-2-bromo-4-(4-nitrophenyl) )-butanoic acid isopropyl ester (prepared by the method of Example 0) during a 5 minute period under a nitrogen atmosphere with stirring. The reaction solution was stirred for 24 hours at room temperature. TLC analysis (solvent system 2) revealed conversion to the monoalkylation title product (Rf=0.78, detection by ninhydrin, iodine and UV activity). The yellow chloroform solution was applied to a 1 inch x 17 inch flash silica gel column that had been pre-eluted with 5% methanolic chloroform. Elution with 300 mL of this solvent system was followed by elution with solvent system 2, yielding fractions containing pure title product which were pooled and evaporated to give 4.85 g (5.46 mmol) of the free base title product as a light oil in 79 % dividend, and further characterized by:

<X>H NMR (CDCl3)

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, 4H), 2.14(m, 1H), 2.00(m, 1H), 1.28(dd, 6H);

<13>C NMR (CDC13)

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 (CDCl3) cm"<1>

3231(N-H), 2978, 2933, 1721 (ester carbonyl), 1601, 1512, 1458, 1345, 1107;

Fast bombardment mass spectrum, m/e 422 [(M+H)] + , 408, 392.

Preparation of end products - ligands

Example 1

Preparation of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, monomethyl, triethyl ester.

In 46 ml of acetonitrile containing 4.6 g (33.3 mmol) of freshly powdered anhydrous potassium carbonate, 700 mg (1.91 mmol) of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane were dissolved 1-acetic acid, 1-methyl ester (prepared by the method in example D). To this slurry was added at once 1.022 g (6.12 mmol) of ethyl bromoacetate. After stirring for four hours at room temperature, the slurry was vacuum filtered, the filter cake was washed with 15 mL of acetonitrile, twice, and the filtrate evaporated in vacuo at 38°C to give the crude title product tetraester as a purple solid. The tetraester was then purified by silica gel chromatography eluting with 5% C 2 H 5 OH/CHCl 3 , then solvent system 3, to give 620 mg (0.988 mmol, 52%) of the desired product. This product was characterized by fast atomic bombardment mass spectrometry ([M+H]<+>=624) and further characterized by:

<X>H NMR (CDCl3)

8.24(d), 7.45(d), 4.94(s), 4.35-4.10(m), 3.79(s), 3.75-l.84(m), 1.34-1.22(m);

<13>C 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 c-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

In 30 ml of 6N HCl was dissolved 300 mg (0.478 mmol) of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraedoic acid, monomethyl, triethyl ester (prepared by the procedure in Example 1) and the mixture was heated at reflux overnight. At the end of this reflux period, the solution was concentrated in vacuo at 70°C to give a yellow solid. This solid was dissolved in 3 mL of water, filtered, and the filtrate evaporated to give 253 mg (0.378 mmol, 79%) of the crude product. This product was purified by preparative TLC [silica gel, developed in 20% NH4OAc/CH3OH (w/v)] and characterized by fast atom bombardment mass spectrometry ([M+H]<+>=526) and further characterized by:

<X>H NMR (D 2 O)

8.21(d), 7.42(d), 4.83(s), 3.83-2.19(m), 1.92(s);

<13>C NMR (D2O)

175.0, 173.6, 171.8, 167.0, 166.3, 146.9, 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 in the tetraacid (prepared by the method in Example 2) was carried out using Adam's catalyst (PtO 2 ) and hydrogen in essentially quantitative yield to give the title product. This product was characterized by fast-atom bombardment ([M+H]<+>=496) and anion-exchange HPLC (on Q-Sepharose™) and further characterized by:

<X>H NMR (D 2 O)

8.12(d), 7.86(d), 5.66(s), 4.73-4.57(m), 4.27-3.78(m), 3.39-2.91 (m);

<13>C NMR (D2O)

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 method of synthesis of this compound was to convert the monoester tetraamine compound from Example D into the monoacid tetraamine compound (6N HCl, refluxed overnight), followed by aqueous alkylation using bromoacetic acid, then reduction of the nitro group to the amine group (PtO2 catalyst) to give a product identical to that described above.

Example 4

Preparation of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid.

In 60 ml of 6N HCl was dissolved 2.0 g (5.48 mmol) of the monoester tetraamine compound (prepared by the method in example D). The mixture was heated to 95°C overnight (about 10 hours) after which it was concentrated in vacuo at 75°C to give 2.53 g (5.11 mmol, 96%) of the monoacid tetraamine tetrahydrochloride as a yellow solid.

The above monoacid tetraamine tetrahydrochloride (0.5 g,

1.0 mmol) was dissolved in 15 ml of water and adjusted to pH 9 with NaOH.

A separate solution of bromine vinegar was prepared (0.71 g,

5.13 mmol) and added to the water solution of the monoacid tetraamine. The pH of the solution was again adjusted to 9 and maintained at this pH during the reaction by small additions of 1.0N NaOH. While the reaction was stirred, aliquots were removed at various times and the progress of the alkylation was monitored by anion exchange HPLC. When the amount of dialkylation product (total of three acetate groups present) had reached a maximum, the entire reaction mixture was freeze-dried to give a dark solid. This crude mixture of alkylated products was then

purified by silica gel chromatography (elution with solvent system 1), followed by preparative anion exchange chromatography (elution with a gradient of 0-100% IM NH4OAc), then by preparative TLC plates (developed in solvent system 4) and finally by silica gel chromatography (elution with solvent system 4). This extensive purification procedure afforded, as a yellow solid, 30 mg (0.06 mmol, 6.0%) of the title product. This product was characterized by fast atom bombardment mass spectrometry ([M+H]<+>=468), anion exchange HPLC and further characterized by:

<X>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 (D2O)

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, 49.29, 47.86, 45.36, 44.26, 42.78, 42.25, 23.72.

Example 5

Preparation of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid pentahydrochloride and α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4, 10-triacetic acid pentahydrochloride.

A crude mixture of alkylated products was prepared by the method described in Example 4. This crude mixture was chromatographed on a silica gel column, eluted with 20% NH 4 OAC/CH 3 OH (w/v). The associated fractions from this column were combined and evaporated to dryness. The crude product with significant amounts of NH4OAC was dissolved in 80 mL of NANOpure™ water and freeze-dried to give a light brown solid

fabric. This solid was dissolved in 20 ml of NANOpure™ water, shaken with PtO2 catalyst under H2 atmosphere until hydrogen uptake stopped. The catalyst was separated by vacuum filtration and the filtrate freeze-dried to give a yellow solid. The solid was dissolved in 3 ml of solvent system 4 and chromatographed on silica gel using solvent system 4. The combined fractions were then evaporated

in vacuo and lyophilized to give 779.6 mg of a pale yellow solid. <13>C NMR and proton NMR suggest that this product is a 50/50 mixture of the two possible geometric isomers. The isomeric mixture was contacted with excess HCl and lyophilized to give 548.4 mg (61% yield) of the title products. Temp.>270°C. Anion exchange chromatography (HPLC system III) showed one significant peak (>94% purity); fast-bombardment mass spectrum showed the geometric isomers [M+H]<+> = 438.

Example 6

Preparation of α-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetramethyl ester.

To a solution of 1.43 g (3.63 mol) of c-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-acetic acid, 1-methyl ester (prepared by the method of example B) in 40 ml of argon-flowed acetonitrile was added 1.71 g (12.34 mmol) of anhydrous potassium carbonate with vigorous stirring. To the reaction mixture was added 1.78 g (11.61 mmol) of methyl bromoacetate, and the reaction mixture was stirred at about 25°C for 48 hours. TLC analysis showed formation of a new product (Rf = 0.62, solvent system 2). To this slurry was added 6.0 g of flash silica gel, and acetonitrile was removed on a rotary evaporator. The resulting material was applied to a 1 inch x 16 inch silica column that had been pre-eluted with 5% methanolic chloroform. About 400 ml of the prepared solution was used to elute several non-polar impurities and unreacted alkylating agent. Then solvent system 2 was applied, the fractions containing the product (Rf = 0.62) were collected, pooled and evaporated to give 2.1 g (3.44 mmol, 95%) of the title product as a pale yellow glass. % NMR indicated that this product must be a 2:1 mixture of conformational or geometrical isomers.

<X>H NMR (CDCl3, 50°C)

8.137(d), 8.128(d), 7.398(d), 7.385(d), 3.82(s), 3.76(s), 3.75(s),3v74(s), 3.68( 8), 1.5-3.52(m);

<13>C NMR (CDC13, 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, 47.5, 45.2, 34.2, 32, 6.

Example 7

Preparation of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetramethyl ester.

In 25 ml of methanol containing 400 mg of 10% palladium-on-carbon catalyst under a nitrogen atmosphere, 1.41 g (2.31 mol) of α-[2-(4-nitrophenyl)ethyl]-1,4 ,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetramethyl ester (prepared by the method in example 6). Excess hydrogen was blown through the solution at one atmosphere for three hours. TLC indicated formation of a strong ninhydrin positive material (Rf = 0.62, solvent system 2). The methanolic catalyst slurry was filtered through celite and evaporation of the solvent afforded the title product, 1.21 g (2.08 mmol, 91%), as a white solid glass (as a 2:1 mixture of conformational isomers). The minor amounts of conformational or geometric isomer were separated from the major isomer by flash chromatography using 10% methanolic chloroform to give the title product as an off-white powder (SP = 89-95°C) and characterized by:

% NMR (CDCl 3 , 50°C)

6.94(d), 6.89(d), 6.69(d), 6.66(d), 3.91(s), 3.80(s), 3.78(8), 3 .74(S), 3.73(S), 3.72(s), 3.71(s), 1.5-3.5(m);

<13>C NMR (CDC1<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, 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;

Fast-bombardment mass spectrum, m/e 602 [M+Na<+>], 618 [M+K<+>]<+>, [624 M+2Na<+->H<+>]<+>.

Example 8

Preparation of α-[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 1.5 g (2.59 mmol) of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4 ,7,10-tetramethyl ester (as the crude 2:1 mixture prepared by the method of Example 7). The reaction mixture was refluxed at about 105°C for 6 hours. TLC (solvent system 1) indicated conversion of the ester (Rf = 0.88) to the title product as the hydrochloric acid salt (Rf = 0.43). Excess solvent was removed on a rotary evaporator and the resulting white solid was dried. The title product as the hydrochloric acid salt was obtained in a yield of 1.6 g and characterized by:

% 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 (D2O, 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, 53.7, 53, 1, 51.6, 47.9, 34.3, 37.6.

Fast-bombardment mass spectrum m/e 524 [M+H]<+>, 546 [M+Na2+]<+>, 568 [M+2Na<+->H<+>]<+>.

Using flash chromatography and solvent system 1, any trace impurities of the ester were removed from the title product as the hydrochloric acid salt. A 1 inch x 23 inch flash silica gel column was loaded with 1.10 g of the title product, as the hydrochloride salt. Fractions containing only the title product, as the mixed potassium ammonium salt, were combined to give 580 mg.

HPLC analysis showed that the material had greater than 98% areal purity at 230 and 254 nanometers.

Fast-bombardment mass spectrum, m/e 562 (M+K)<+>, 584 [M+K+-Na+-H+] , 600 [M+2K+-H+]"1".

Example 9

Isolation of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid, mixed potassium ammonium salt.

A crude solution containing both the tetra- and triacids (2.05 g)

(prepared by the procedure of Example 8) was dissolved in a minimum amount of solvent system 1 and applied to a 12 inch x 3 inch column of flash silica gel which had been pre-eluted with this solvent. Elution of fractions containing the title product (Rf = 0.63 in solvent system 1) was collected, yielding 200 mg of c-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4 ,10-triacetic acid, mixed potassium ammonium salt. ^H and <13>C NMR analysis suggested that this triacid was the symmetrical isomer (the 1,4,10-triacid positional isomer):

^■H NMR (D2O, pD = 0.5 with DC1, T = 90°C)

7.52(d), 7.46(d), 3.60(m), 3.54(m), 3.19(m);

<13>C NMR (D2O, 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, 51, 2, 31,2.

Fast-bombardment mass spectrum, m/e 466[M+H<+>]<+>, 488[M+Na<+>]<+>, 504[M+K<+>]<+>, 526[ M+K<+>+Na<+->H<+>]<+>, 542[M+2K-H)]<+.>

Example 10

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

Methyl bromoacetate (565 µl, 5.97 mmol) was added to a stirred mixture of α-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane, 1-acetic acid, methyl ester (580 mg, 1, 47 mmol)

(prepared by the procedure in Example N) and powdered potassium carbonate (1.15 g, 8.32 mmol) in acetonitrile (20 mL). The reaction mixture was stirred at room temperature for 2 hours. The mixture was then filtered and the filtrate was concentrated to an oil in vacuo. The crude product was purified by chromatography in (silica gel, 8% methanol in methylene chloride). Title-

the product was obtained as a yellow solid (469 mg, 52%), TLC Rf = 0.4 (silica gel, 10% CH3OH in CHCl3), and further characterized by:

<13>C NMR (CDCl3)

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-tetraazacyclododecane-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, tetramethyl ester (prepared by the procedure of Example 10) in concentrated HCl (5 ml , J.T. Baker ULTREX) was refluxed under a nitrogen atmosphere for 5 h. The solution was then concentrated to dryness in vacuo to leave a solid residue. This was purified by chromatography (silica gel, solvent system 6) to give the title product as an off-white solid

(209 mg). This substance was converted to the tetrahydrochloride salt and was characterized by:

<13>C NMR (D20-DC1, 80°C)

171.0, 166.9, 161.2, 139.0, 125.5, 119.3, 110.7, 56.8, 54.5, 52.7, 51.0, 49.2, 49, 0, 46.3, 42.9.

Example 12

Preparation of α-(2-methoxy-5-aminophenyl)1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, tetraammonium salt.

To a solution of α-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (157 mg) (prepared by the procedure in Example 11) in water ( 20 ml) was added platinum oxide (20 mg). This mixture was hydrogenated (1 atmosphere of hydrogen) for 1 hour at room temperature. After filtration, the material was further purified by chromatography (silica gel, solvent system 5) to give the title compound (141 mg) as an off-white solid, and characterized by:

<13>C NMR (D20-DC1)

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.

To 4 ml of argon-flowed acetonitrile with stirring was added 100 mg (0.236 mmol) of 1-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane, the tetrahydrochloride salt (prepared by the procedure in Example J), 260 mg (1, 88 mmol) of potassium carbonate, and 108 mg (0.708 mmol) of methyl bromoacetate. The mixture was stirred for 48 hours at 25°C. The saline solution was applied

a 1 cm x 4 cm flash silica gel column and was 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:

1-H NMR (CDCl 3 )

7.29 (d), 6.75 (d), 4.62 (s), 4.20 (wide s), 3.70 (s),

3.69 (s), 3.64 (s), 3.62 (t), 3.27 (s), 3.06 (t), 2.82 (wide t), 2.79 (wide t) ;

<13>C NMR (CDCl3, 75 MHZ),

171.4, 171.0, 148.4, 132.8, 117.5, 115.0, 55.9, 55.3,

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

Example 14

Preparation of 1-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, hydrochloride salt.

In 2 ml of 6N hydrochloric acid, 42 mg (0.085 mmol) of 1-(4-amino-benzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, 4,7,10-trimethyl ester ( prepared by the method in example 13) was heated to 80°C for 2 hours. TLC indicated several products (Rf = 0.60 for the desired title product, solvent system 1). Removal of solvent gave 41 mg of the crude title product.

Example 15

Preparation of 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, 4,7,10-trimethyl ester.

To a reading 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 argon-flowed acetonitrile was added with vigorous stirring 3.17 g of anhydrous potassium carbonate. To this reaction mixture was added dropwise over a period of 5 min. period 3.20 g (20.9 mmol) methyl bromoacetate. The reaction mixture was stirred for 17 hours under an argon atmosphere. TLC analysis showed conversion of the starting material (Rf = 0.73, solvent system 1) to a new product (Rf = 0.46, solvent system 1). To the solution was added 70 ml of chloroform, and the solution with the slurried salt was applied to a 1 inch x 7 inch flash silica column. The product was eluted using 10% methanol in chloroform to give 2.95 g of the title product as an amber oil which formed a brittle glass on vacuum drying (78%). The title product was characterized by:

1 H NMR (CDCl 3 )

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

<13>C NMR (CDCl3, 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 1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetra-azacyclododecane-4,7,10-triacetic acid, 4,7,10-trimethyl ester.

In 50 ml of methanol were dissolved 2.8 g (5.18 mmol) of crude 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, 4 ,7,10-trimethyl ester (prepared by the method in example 15). To the stirred solution which was purged with nitrogen, 10% palladium on carbon catalyst (600 mg) was added. The solution was kept under an atmosphere of nitrogen, and then hydrogen (1 atm. 20-25°C) was blown through the stirred solution for 2.5 hours. The solution was then purged for several minutes with the nitro, and the catalyst removed by filtration through a thin layer of celite. TLC analysis (10% methanol in chloroform) revealed the title product (Rf =

0.14). The title product was eluted with chloroform from silica gel to give 2.2 g (4.33 mmol, 83%) as a white glass and characterized by:

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 (CDCl3, 75 MHz)

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

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

Fast atomic bombardment mass spectrum, m/e 508 [M+H<+>]<+.>

Example 17

Preparation of 1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, hydrochloride salt; (EA-D03A).

In 30 ml conc. HCl was dissolved 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 by the procedure in Example 16). The solution was stirred at 70-80°C for 2.5 hours, and the solution was allowed to cool with stirring to 25°C, and stirring was continued for about 18 hours. The solvent was removed on a rotary evaporator at reduced pressure (10 mm, 75°C).

Solvate and excess hydrogen chloride were removed from the resulting clear oil by vacuum drying (10-<1> mm, 45°C). The title product was provided as a white solid, in a yield of 890 mg, (Rf = 0.46, solvent system 2) and further characterized by:

^■H NMR (D 2 O, pD = 1.9, T = 90°C)

7.50(d), 7.41(d), 4.26(s), 3 .43-3.68(m) , 3.0-3.3(m);

<13>C NMR (D2O, 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;

Fast-bombardment mass spectrum, m/e 466 (M+H+) , 488 [M+Na<+>], 510 [M+2Na<+->H<+>]<+>.

HPLC analysis showed greater than 92% areal purity (254 nm) using a 10 cm Partisil-5-OD53 RAC II reverse phase column. The eluent was 10% acetoniril in 0.05 M pH = 7.0 potassium phosphate solution.

Example 18

Preparation of 1-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid; (SCN-EA-D03A).

1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, hydrochloride salt (prepared by the method of Example 17, 106 mg, 0.21 mmol) was dissolved in 2 ml of water under a nitrogen atmosphere with stirring. Sodium bicarbonate (105.6 mg, 1.26 mmol) was slowly added to prevent foaming by carbon dioxide evolution (resulting pH =

8.0). Thiophosgene (16.8 µl, 0.22 mmol) was added and after one hour of vigorous stirring, TLC analysis (20% water in acetonitrile) indicated conversion of starting material (Rf = 0.12) 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 substance (KBr pellet) considered the presence of the isothiocyanate group (SCN = 2150 cm-<1>).

Example 19

Preparation of 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, triethyl ester.

To 5 ml of a stirred acetonitrile solution containing

395 mg (1.12 mmol) of 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane (prepared by the procedure in Example G) and 1.5 g (11 mmol) of K 2 CO 3 was added 496 µl (4.5 mmol) of ethyl bromoacetate in one portion. After heating at 68°C under a N 2 atmosphere for one hour, the resulting slurry was filtered. The filter cake was washed with CH 3 CN (2 x 10 mL). The filtrate was then concentrated to give the crude product

as a viscous oil, triturated with 30 mL of diethyl ether, and concentrated to give 650 mg (100% yield) of l-[2-(4-nitrophenyl)ethyl]-

1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, triethyl ester and characterized by:

% NMR (CDCl3)

8.2(d), 7.6(d), 4.3(q), 2.6-3.8(m), 155(t);

Fast-bombardment mass spectrum [M+H]<+> = 588.

Example 20

Preparation of 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid.

To a slurry 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 by the method in example 19) in 15 ml of water was added 59 pl of NaOH (50% w/w, 3 eq.). The mixture was stirred at 80°C for 6 hours. The resulting homogeneous orange solution was then cooled to room temperature and lyophilized to give a brown solid which was purified by HPLC (Q-Sepharose™, HPLC system III) using a linear gradient of 0-10% aqueous HOAc over of 60 min. to give (50%) of the hydrolysed product 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, and characterized by:

<X>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-tetraazacyclododecane-4,7,10-triacetic acid; (EA-D03A).

In a Parr hydrogenator was placed 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-triacetic acid (prepared by the method in example 20) and 40 mg of 10% Pd/C. The slurry was shaken until hydrogen absorption stopped. After filtration, the aqueous solution was freeze-dried to give 197 mg (98%) as a light brown solid, 1-[2-(4-aminophenyl)-ethyl]-1,4,7,10-tetraazacyclododecane-4,7, 10-triacetic acid, and characterized by:

<1->H NMR (D2O)

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

<13>C NMR (D2O)

173.85, 172.50, 137.90, 131.90, 130.22, 121.55, 56.25, 55.14, 54.42, 50.12, 49.65, 49.31, 31, 00.

Example 22

Preparation of 1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, 4,7,10-trimethyl ester.

To a stirred acetonitrile solution (20 mL) containing

1.0 g of 1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane (3 mmol) (prepared by the procedure in Example E) was added to 1.6 g of methyl bromoacetate (11 mmol) in a portion. After one hour potassium carbonate (0.3 g) was added and stirring continued at room temperature for 12 hours. The reaction mixture was then filtered, and the filtrate concentrated in vacuo. The resulting residue was column chromatographed (silica, CH3CN/CH3OH, 9:1, V:V) giving the triester as a white solid in 68% yield after concentration, and was further characterized by:

<1->H NMR (CDCl3)

8.19(d), 8.14(s), 7.18(d), 2.58-3.99(m);

<13>C NMR (CDCl3)

173.53, 172.83, 164.59, 142.28, 128.51, 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-tetraazacyclododecane-4,7,10-triacetic acid.

A 6M hydrochloric acid solution (3 ml) containing 0.25 g of 1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, 4,7,10-trimethyl ester (0.45 mmol) (prepared by the method of Example 22) was stirred and heated at 100°C for 24 hours. After cooling to room temperature, 5 ml of water was added and the aqueous solution freeze-dried to give a light brown solid. After column chromatography (silica, solvent system 5), the triacid was isolated in 60% yield as an almost white solid, SP = 228-230°C (dec), and further characterized by:

<X>H NMR (D 2 O)

8.18(d), 8.09(s), 7.12(d), 3.87(s), 2.10-3.60(m);

<13>C NMR (D2O)

180.45, 179.80, 163.66, 140.22, 128.60, 126.24,

122.6, 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-tetraazacyclododecane-4,7,10-triacetic acid.

To a nitrogen-flushed aqueous (50 ml) solution containing 50 mg of PtO2 was added 50 mg of 1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (prepared by the procedure in example 23). After hydrogenation in a Parr bomb for one hour, the solution was filtered and the aqueous filtrate freeze-dried to give the aniline derivative as a light brown solid (95% yield), MP>240°C dec, and further characterized by:

3-H NMR (D 2 O)

7.54(bs), 7.21(d), 7.09(d), 7.05(s), 6.88(8), 3.84(s), 3 .78(8) , 2 .85-3.56m) ;

<13>C NMR (D2O)

180.59, 179.77, 153.38, 124.16, 124.03, 122.82,

120.18, 113.68, 112.43, 59.06, 57.02, 55.96, 53.67, 50.52, 50.08, 49.70, 49.04, 48.32.

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-nitrobenzyl-1,4,7,10-tetraazacyclododecane (200 mg, 0.62 mmol)) (prepared by the procedure in Example F) was added bromoacetic acid

(309 mg, 2.2 mmol), and NaOH (3.5 mL, ∼1 M) with stirring at room temperature. The progress of the reaction was checked by LC (anion exchange, Q-Sepharose™) and the pH was maintained at about 8 by addition of NaOH as needed. After 12 hours, the solution was freeze-dried and the solid residue chromatographed (column) on silica gel eluting with solvent system 5. The major yellow fraction was concentrated, dissolved in H 2 O and freeze-dried to give the desired product as a pale yellow powder (150 mg, 49%); SP = 230-235°C (dec), and further characterized:

<13>C NMR (D2O)

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,lo-tet raazacyclododecane-4,7,10-triacetic acid.

To an argon-purged aqueous solution (25 mL) of N-(2-hydroxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (200 mg, 0.4 mmol) (prepared by the procedure in example 25) PtO 2 (130 mg) was added followed by the introduction of H 2 (Parr hydrogenator). After complete disappearance of the yellow color, the solution was flushed with argon and filtered. The aqueous solution was then freeze-dried to give the product as a light brown solid (146 mg, 78%).

Example 27

Preparation of α-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic) acid, 1-isopropyl-4,7,10-trimethyl ester; (PN-DOTMA trimethylisopropyl ester).

To 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 by the method in example P) in 35 ml of dry acetonitrile was added 1.15 g (8.32 mmol) of anhydrous potassium carbonate with vigorous stirring under nitrogen. Optically active α-benzenesulfonate of 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. TLC analysis indicated formation of new products (Rf=0.71, solvent system 2 for the title tetraesters and Rf=0.89 for triesters). The resulting solution of slurried 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 1 inch x 16 inch flash silica gel column using solvent system 2 as eluent to give the title product as an undissolved mixture of tetraester diastereomers (1, 00 g, 1.47 mmol, 62% yield) which was free of underalkylated products.

^■H NMR analysis of this substance showed that it contained about 0.5 equivalents of unreacted benzenesulfonate derivative. NMR spectra of this substance were non-coalising at 60°C in chloroform:

<X>H 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);

IR (CDCl3) cm"<1>

2980, 2840, 1725, 1710 (ester carbonyl), 1590, 1510, 1440, 1340;

Fast atomic bombardment mass spectrum, m/e 702 [M+Na<+>]<+>, 687.

Example 28

Preparation of o-[2-(4-aminophenyl)ethyl]-1,4,7,10-[tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic) acid , 1-isopropyl-4,7,10-trimethyl ester; (PA-DOTMA trimethylisopropyl ester).

α-[2-(4-nitrphenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic) acid, 1- isopropyl-4,7,10-trimethyl ester (950 mg, 1.40 mmol) uncorrected) (prepared by the method of Example 27), which contained unreacted o-[3-(4-nitrophenyl)propyl]-1,4,7 ,10-tetraazacyclododecane-1-acetic acid, 1-methyl ester, was dissolved in 10 ml of 30% aqueous methanol containing

1.00 g of 10% palladium on carbon catalyst under a nitrogen atmosphere. Excess hydrogen was blown through the solution for 7 hours. At this point, TLC inspection revealed formation of a strong ninhydrin positive material (Rf=0.62 solvent system 2, Rf=0.71 for starting material). The methanolic catalyst slurry was filtered through celite and evaporation of solvent gave 800 mg of the crude title product as a clear oil containing benzenesulfonic acid (Rf = 0.09 solvent system 2) from the accompanying hydrogenolysis of the benzenesulfonate ester. The crude oil was chromatographed on a 1 inch x 8 inch flash silica gel column with 10% methanol in chloroform eluting a bright yellow band in the pore volume. Solvent system 2 was then applied to elute the title product (480 mg) in 52% yield as a mixture of unresolved diastereomers and geometric isomers. Three distinct ab quartets were observed in the aromatic region by ^-H NMR:

<1>H NMR (CDCl 3 , 30°C)

6.63-8.0 (m (quartets), 4H), 5.07(m, 1H, isopropylmethine H), 3.53-3.9(m, 13 H with four singlets at 3.85, 3, 73, 3.69

and 3.56), 3.46(m, 1H), 3.25(broad, 3H), 2-3.1(m, 14H), 1.0-2.0(m, 15H;

<13>C NMR (CDCl3, 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, 52.9, 52.7, 52.1, 50.8, 50.7, 50.6, 47.3, 47.1, 47, 0, 46.9, 46.5, 46.3, 46.1, 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 (CDC123) cm"<1>

2960, 2840, 1720, 1510, 1450, 1375;

Fast-bombardment mass spectrum, m/e 672 [M+Na<+>]<+>, 686.

Example 29

Preparation of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic) acid;

(PA-DOTMA).

The diastereomeric mixture of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(R-methylacetic) acid, l-isopropyl-4,7,10-trimethyl ester (400 mg,

0.59 mmol) (prepared by the method in Example 28) was dissolved in 50 ml of 6N concentrated hydrochloric acid and subjected to reflux for 30 hours. TLC analysis (solvent system 1) indicated conversion of ester (Rf=0.98 solvent system 1) to two new spots (Rf=0.6, high diastereomer and Rf=0.54, low diastereomer solvent system 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) was obtained as the hydrochloride salt. Preparative separation of the two diastereomers of the title product was accomplished by loading 290 mg (0.51 mmol) of the crude salt onto a 1 x 13 cm silica flash gel column eluted with solvent system 8. The high Rf diastereomer (119 mg, 0.21 mmol) was obtained in purer form than the low Rf diastereomer (90 mg, 0.16 mmol) since the high Rf diastereomer eluted from the column first:

^•H NMR for high Rf diastereomer:

<X>H NMR (D 2 O, 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, 19H) , l, 89(m, 1H), 1.37(m, 1H), 1.15(d, 3H), 1.10(d, 3H), 1.06(d, 3H);

<13>C NMR (D2O, 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, 57, 4, 54.1, 49.7, 49.6, 48.2, 47.7, 47.2, 34.8, 33.8, 9.6, 9.1, 9.0;

Fast atomic bombardment mass spectrum, m/e 566 [M+H<+>]<+>,

588 [M+Na<+>]<+>, 604 [M+K<+>]<+>, 626 [M+K<+>+Na<+->H<+>]<+>, 642 [M+2K<+->H<+>]<+>.

Production of end products - Complexes

Metal ligand complexes were prepared by various procedures as shown below. The procedures included mixing the metal and the ligand in aqueous solution, and adjusting the pH to the desired value. The complexation was carried out in solutions containing salts and/or buffers as well as water. Occasionally it was found that heated solutions gave higher complex yields than when the complexation was carried out at ambient temperatures. The work-up used in the synthesis of BFC has also been shown to affect the complexation.

Example 30

Preparation of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, ammonium salt, samarium(III) complex; Sm(BA-DOTA).

A small sample, 53 mg (0.094 mmol) of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared by the procedure of Example 3) was dissolved in 100 pl of water and treated with 3 ml of 0.043M Sm(OAc)3 solution (48.8 mg,

0.129 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 complexation was complete by anion exchange chromatography, HPLC system III, the solution of the complex was cooled to room temperature (about 25°C) and freeze-dried. The samarium complex was purified by silica gel chromatography (elution with solvent system 1). This progress yielded the title product in 82% yield. This complex was characterized by TLC, fast atom bombardment mass spectrometry, anion exchange HPLC, and further characterized by:

<X>H (D20)

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 (D2O)

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, 49.7, 45.5, 43.5, 23.6.

Fast-bombardment mass spectrum, [M+H<+>]<+> = 645 (Sm isotope sample).

Example 31

Preparation of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium(III) complex.

A solution of 150 µl Sm-153 (3 10~<4>M, in 0.IN HCl) and

600 µl of SmCl3 (3 x 10"<4>M) in 0.1N HCl was prepared by adding the Sm-153 solution to the SmCl3 solution. Then 5.0 µl of 2.OM NaOAc was added followed by 45 µl of 50 mM ligand (prepared by the procedure of Example 3) in HEPES buffer The pH of the solution was adjusted to 7 with 275 µl of 0.5 M HEPES.

This solution was divided into two fractions of 500 µl each, and one fraction was heated for 1 hour at 100°C.

The solutions were passed through SP-Sephadex™ cation exchange resin. The percentage of metal complexed was determined using the method described previously. The results showed 99.6% of the metal as complex for the heated sample, and 96.6%

for the unheated sample.

Stability of chelates by pH profile.

The stability of metal ligand complexes was measured by subjecting them to different pH values and analyzing for complex in solution. At low pH, protonation of the ligand can cause release of metal. At high pH, the metal hydroxides compete with the chelation. Therefore, when searching for inert chelates, a desirable inert chelate will remain as a chelate when exposed to high and low pH values.

Profiles of the inertness at different pH values for some of the chelates of this invention were prepared using the methods described in the following examples.

In some cases, non-chelated metal was removed by passing the solution through a cation exchange resin. Dilute sodium hydroxide and hydrochloric acid solutions were used to adjust the pH

for the complex solutions from about 1 to about 14.

Percent metal as complex was then determined by the methods described earlier.

In a similar manner, several bifunctional chelate conjugates were prepared and subjected to pH 2.8, 4.0 and 6.0. The amount of conjugate remaining in solution was determined by HPLC using a radiometric detector. The results are shown in Example XX

in the biology department.

Example 32

The pH stability of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium(III) complex.

The heated and unheated samples from Example 30 were all divided into 2 x 250 µl aliquots. A 250 µl aliquot was adjusted with HCl to give the pH values shown in the following table. The other 250 µl aliquot was adjusted with µl amounts of NaOH to the desired pH values. When the desired pH was reached, a 25 µl aliquot was removed and the percent metal complexed determined as previously described. The results are as follows:

These data show the stability of this sample whether it is heated or not, and regardless of the tested pH.

Example 3 3

Preparation of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, yttrium(III) complex.

10 µl of a solution containing 1.4 mg/100 µl of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared by the method in example 3) was added to

100 µl of Y(0Ac)3 (0.003M) added to Y-90. To this solution was added 500 µl of water followed by 125 µl of NaOAc (0.5M). Water (265 µl) was added to the solution to bring the total volume to 1 mL. This solution was divided into two aliquots. One aliquot was heated to 100°C for 1 hour. Both heated and unheated samples were tested to determine percent metal complexed using the cation exchange method described previously. The results showed 84% and 4% for the metal as a complex for respectively. the heated sample and the non-heated sample.

Example 34

Preparation of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, lutetium(III) complex.

A solution of α-4-(-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared by the method of Example 3) was dissolved in distilled water to give a concentration of 1 .63 mg/ml.

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

A volume of 30 µl of 0.01 M lutetium solution was added

2 pl Lu-177 solution. The appropriate amount of ligand from above was added, and HEPES buffer (0.1M, pH = 7.6) was added to give a total volume of 1.0 ml. The resulting solutions were 0.3 mmol in ligand and lutetium. The solution was then heated to 100°C for 1 hour and the percentage of Lu as complex determined to be 86% by the cation exchange method described earlier.

Example 35

Preparation of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium(III) complex.

A sample, 7 mg (10.8 pmol) of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium(III) complex (prepared by the method in example 30) was dissolved in 400 µl of water. Excess thiophosgene (50 µl) was added, followed by 400 µl CHCl 3 , and the two-phase reaction stirred vigorously for 30 min. At the end of this time, the aqueous layer was extracted with 500 µl CHCl 3 four times, and the aqueous layer was then lyophilized to give the desired title product in quantitative yield.

UV showed that this compound had a band at 272 and 282 nm. TLC, silica gel developed at 75:25 V:V CH 3 CN:H 2 O gave Rf = 0.38. The starting material had an Rf = 0.19. IR showed -SCN stretch at 2100 cm"<1>; fast-bombardment mass spectrum [M+H<+>]<+> = 687.

Example 36

Preparation of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, ammonium salt, yttrium(III) complex.

A sample of 90 mg (160 mmol) of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared by the procedure in Example 3) was dissolved in 1 ml water. To this solution was added 3 ml of water containing 51 mg (192 mmol) of Y(0Ac)3. The reaction mixture, pH = 6 to 7, was then heated at 100°C for two hours. The resulting solution was then passed through glass wool and lyophilized to give 126 mg of yellow solid. The solid was chromatographed on silica gel (solvent system 1) to give 71 mg (76%) of the desired complex product. Analysis by anion exchange showed the same retention time as found for the analogous Sm complex. The title product was characterized by:

<13>C NMR (D2O)

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;

<X>H NMR (D 2 O)

6.90(d), 6.70(d), 4.40(s), 3.45-3.06(m), 2.71-2.10(m), l.75(s) .

Fast-bombardment mass spectrum, [M + H<+>]<+> = 582.

Example 37

Preparation of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, sodium salt, yttrium(III) complex.

A sample of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the yttrium complex (prepared by the procedure of Example 36) (10 mg, 17 pmol) was dissolved in 400 pl H20. To this solution was added 64 µl of thiophosgene (excess) and 400 µl of CHCl 3 , and the resulting mixture was stirred vigorously for 40 min. During this time, several small additions of solid NaHCO 3 were made to maintain the pH

of about 8. At the end of the reaction, the aqueous layer was separated and extracted with 1 ml of CHCl 3 , four times, and lyophilized. The title product was characterized by TLC and UV spectroscopy.

Example 38

Preparation of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, yttrium(III) complex, ammonium salt; NH 4 [Y(PA-DOTA)].

The ligand α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared by the procedure in Example 8) was converted to the mixed protonated ammonium salt by chromatography on a strong cation exchange resin (SP-Sephadex™ C-25, Pharmacia). The column was in the protonated form and was washed with distilled water. A concentrated aqueous solution of the ligand was applied to the column, followed by washing the column with distilled water. The ligand was eluted from the column with 0.5M NH 4 OH. The eluent was reduced to dryness.

A solution of Y(OAc)3.4H2O (0.0728 g, 0.215 mmol) in 5 mL of distilled water was added to a hot solution of the mixed protonated ammonium salt ligand (0.120 g, 0.215 mmol) in 5 mL of water. The solution was brought to reflux, and the pH adjusted to about 7.0 with 0.1M NH 4 OH. After refluxing for 1 hour, the solution was cooled and reduced to dryness. Excess NH40Ac was removed by heating the white solid in an oil bath at about 105°C under vacuum. The yield of the title product (which contained about one equivalent of ammonium acetate) was 0.142 g (94%) and was characterized by:

13C NMR (D2O, 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;

Fast-bombardment mass spectrum, m/e 610 (positive ion, [M"+2H<+>]<+>), 608 (negative ion, M").

Example 39

Preparation of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium(III) complex, ammonium salt; NH 4 [Sm(PA-DOTA)].

When the procedure of Example 38 was repeated, using Sm(OAc)3.3H20 (0.082 g, 0.215 mmol) instead of Y(OAc)3.4H20,

the title product (which contained about one equivalent of ammonium acetate) was prepared in a yield of 0.155 g (94%) and was characterized by:

1<3>C NMR (<D>20, 88°C, 75 MHz)

23.5, 27.2, 35.6, 50.5, 54.5, 56.0, 57.2, 57.9,

69.5, 71.5, 122.3, 133.0, 139.7, 181.2, 188.4, 189.3, 190.9;

Fast-bombardment mass spectrum, m/e 673 (positive ion, [M~+2H<+>]<+>, Sm isotopic pattern), m/e 671 (negative ion, M~, Sm isotopic pattern).

Example 4 0

Preparation of α-[2-(4-isothiocyanatophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium(III) complex, ammonium salt; NH 4 [Sm(SCN-PA-DOTA)].

Solutions of Sm(OAc)3.3H2O (27.9 mg, 381.56 g/mol,

73.1 pmol) in 10 ml of distilled water and 42 mg of PA-DOTA, mixed potassium ammonium salt (632.97 g/mol, 66.4 pmol) (prepared by the procedure of Example 8) in 10 ml of distilled water were mixed and heated in a steam bath. After 30 min. the reaction to form [Sm(PA-DOTA)] was complete as determined by HPLC using the procedure described in Example 41. The solution was reacted with a solution of 45.4 mg CSC12 (114.98 g/mol, 395 jjmol)

in 20 ml of chloroform by shaking in a separatory funnel. The turnover was complete after about 1 min. as determined by HPLC and in the absence of a positive test with ninhydrin (0.2% in ethanol) when the aqueous solution was spotted on a silica gel TLC plate (the starting chelate, [Sm(PA-DOTA)], tested positive). The chloroform layer was removed and the aqueous layer was washed with two 20 mL portions of chloroform. The aqueous layer was reduced to dryness by the addition of 100 mL of acetonitrile and evaporated at ambient temperature under a stream of nitrogen to give 56 mg of the title product.

Fast-bombardment mass spectrum, m/e 715 (positive ion, [M~+2H<+>]<+>, Sm isotopic pattern), m/e 713 (negative ion, M", Sm isotopic pattern).

Example 41

HPLC analysis of the chelation rate of Y<3+> with PA-DOTA.

The rate of the PA-DOTA chelation with Y<3+> was studied as a function of the workup used in the synthesis of PA-DOTA. The degree of chelation was checked by HPLC using an Alltech Econosphere™ C18 100 mm column. The gradient used was: A) 95:5 with pH 6.0, 0.05M NaOAc buffer:CH3CN,

B) 30:70 with pH 6.0, 0.05M NaOAc buffer:CH3CN, A to B on

15 min.. A solution of PA-DOTA, hydrochloride salt, (retention time = 1.31 min.) and of PA-DOTA mixed potassium ammonium salt (retention time = 4.55 min.)" both prepared by the method of Example 8, was prepared as 1.6 mM, pH 6.0 0.5 M NaOAc buffer. One ml (1.6 pmol) of each of the two solutions was reacted with 0.2 ml (8 pmol) of Y(OAc) 3.4H 2 O solution (40 mM in pH 6.0 0.5M NaOAc buffer). The chelation was determined by the appearance of a peak at a retention time = 3.96 min. (confirmed by comparison with a sample from Example 38). The results of the chelation were as follows.

Obviously, the method of purification of BFC has an effect on the rate of chelation.

Example 42

Preparation of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic-4,7,10-tris-(methylacetic)acid, samarium ( III) complex, ammonium salt; NH 4 [Sm(PA-DOTMA)].

A solution of Sm(OAc)3.3H2O (13.2 mg in 2 mL of distilled water) was added to a solution of ot-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1 -(R,S)-acetic-4,7,10-tris-(methylacetic) acid (30 mg in 2 ml of distilled water), (prepared by the procedure of Example 29, high Rf diastereomer). The solution with pH 6.5 was heated on a steam bath, and the conversion was checked by HPLC. After 30 min. heating, the solution was reduced to dryness on a rotary evaporator and dried in a vacuum oven.

Fast-bombardment mass spectrum, m/e 715 (positive ion, [M+2H<+>]<+>, Sm isotopic pattern), 737 (positive ion, [M+H<+>+Na<+>]<+ >, Sm isotopic pattern) 713 (negative ion, (M~), Sm isotopic pattern).

In the following examples, the complex solutions were passed through an ion exchange column to remove any excess free metal from the solution. Labile systems would return to equilibrium, and similar amounts of the free metal would be in solution. Inert systems would not return to equilibrium and the amount of non-chelated metal would decrease.

Thus, although a complex can be formed in low yield, purification by passing it through an ion exchange resin can result in a usable stable complex with no uncomplexed metal.

Example 43

Preparation of α-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium(III) complex.

A 3 x 10~<2>M solution of α-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared by the method in Example 12) was prepared by dissolving 5.8 mg in 325 µl of NANOpure™ water. A 10 µl aliquot of this solution was added to 990 µl of 3 x 10"<4>M SmCl3 (in 0.1N HCl) spiked with Sm-153. The pH was then brought to 7.0 with NaOH. This solution was split into two fractions of 500 µl each, and one fraction was heated for 1 hour at 100° C. Both heated and unheated samples were tested to determine the percent metal complexed by the cation exchange procedure described earlier. The solution was then passed through SP-Sephadex ™ resin. The complexation was again determined as in the purified samples. The results are shown in the following table.

Example 44

pH stability of α-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid, samarium(III) complex.

The heated and unheated purified samples from Example 43 were all split into 2 x 250 µl aliquots. One aliquot was adjusted with HCl and the other with NaOH to give the pH values shown in the following table. After the desired pH had been achieved, the sample was allowed to stand for 10 min., and the amount of metal as a complex was determined by the cation exchange method described earlier. The results are shown in the following table.

Example 45

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

A 3 x 10~<2>M solution of α-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (prepared by the method in Example 11) was prepared by dissolving 7.9 mg in 464 µl of NANOpure™ water. A 10 µl aliquot of this solution was added to 950 µl of 3 x 10"<4>M SmCl3 (in 0.1N

HCl) added with Sm-153. The pH was then brought to 7.0 with NaOH. This solution was split into two fractions of 500 µl each, and one fraction was heated for 1 hour at 100°C. Both heated and unheated samples were tested to determine the percent metal complexed using the cation exchange method described previously. The solutions were passed through SP-Sephadex™ resin. Complexation was then determined on the purified samples using the general procedure described hereinbefore. The results are shown in the following table:

Example 46

pH stability of α-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid, Sm(III) complex.

The heated and unheated purified sample from Example 45 were both divided into two aliquots. One aliquot was adjusted with dilute HCl and the other with NaOH to give the pH values shown in the following table. After the desired pH values had been achieved, the sample was allowed to stand for 10 min., and the amount of metal complexed was determined by the cation exchange method described earlier. The results are shown in the following table:

Example 47

Preparation of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, samarium(III) complex.

A 0.0219 M (100 µl, 2.195 mmol) solution of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (prepared by the method of Example 4) was contacted with a 0.043 M

(25 µl, 1.075 mmol) solution of Sm(OAc) 3 . The reaction mixture was subjected to anion-exchange HPLC (Q-Sepharose™ column, 1 cm x 25 cm, flow rate = 2 ml/min, eluted with 0-1 M NH 4 OAc gradient over 30 min). After 1 hour, the complex (retention time = 11.1 min.) had formed in 77% yield (area percentage), and was completely dissolved from the ligand peak (retention time = 15.5 min.). Further probability that

the title complex had formed was obtained by silica gel TLC (solvent system 4) whereby the ligand had an Rf =

0.59 and the complex had an Rf = 0.00.

Example 48

Preparation of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, samarium(III) complex and α-(4-aminophenyl)-1,4,7,10 -tetraazacyclododecane-1,4,10-triacetic acid, samarium(III) complex.

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

Example 49

Preparation of c-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, samarium-153 complex and α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane -1,4,10-triacetic acid, samarium-153 complex.

A 3 x 10"<2>M solution of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid pentahydrochloride and c-(4-aminophenyl)-1,4, 7,10-Tetraazacyclododecane-1,4,10-triacetic acid pentachloride (prepared by the procedure of Example 5) was prepared 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~<2>M SmCl3 (in 0.1N HCl) spiked with Sm-153 The volume was brought to

1 ml by adding water. The pH was then brought to 7.0 with NaOH. This solution was split into two factions on

500 µl each, and one fraction was heated for 1 hour at 100°C.

Both heated and unheated samples were tested to determine the percent metal complexed by the cation exchange method described earlier. The results showed 89% and 96% of the metal as complex for, respectively. the heated and unheated samples.

Example 50

The pH stability of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, samarium (III) complex, and α-(4-aminophenyl)-1,4, 7,10-Tetraazacyclododecane-1,4,10-triacetic acid, samarium(III) complex.

The heated sample from Example 49 and the unheated sample were both divided into 2 aliquots. One aliquot was adjusted with HCl and the other with NaOH to give the pH values shown in the following table. When the desired pH had been achieved, the samples were allowed to stand for 10 min. and the percent metal complexed was determined by the cation exchange method described earlier. The results are shown in the following table:

Example 51

Preparation of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, samarium(III) complex.

13 microliters of 2 x 10~<2>M solution of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (prepared by the procedure in Example 4) 15 µl of Sm Cl3 (0.02M 0.1N HCl)

and 2 µl of Sm-153, the volume was brought up to 1 ml with NANOpure™ water. The pH was adjusted to 7.0 with NaOH. This solution was split into two fractions of 500 µl each, and one fraction was heated for 1 hour at 100°C. Both heated and unheated samples were tested to determine the percent metal complexed using the cation exchange method described previously. The results showed 74% and 42% of the metal as complex for, respectively. the heated and unheated samples.

Example 52

The pH stability of α-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, samarium(III) complex.

The heated sample from Example 51 was divided into 2 aliquots. One aliquot was adjusted with HCl and the other with NaOH to give the pH values shown in the following table. After the desired pH had been reached, the sample was allowed to stand for 10 min. and the amount of metal as a complex was determined by the cation exchange method described earlier. The results are shown in the following table.

Example 53

Preparation of 1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, lutetium(III) complex.

A solution of EA-D03A (prepared by the method of Example 17) was made by dissolving the solid in water. The final concentration was 0.0IM.

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

A volume of 30 µl of 0.01M Lu solution was added to 2 µl of Lu-177 solution. A volume of 30 µl of ligand was added, and HEPES buffer (0.1 M, pH = 7.6) was added to give a total volume of 1.0 ml. The resulting solution was 0.3 mM in ligand and lutetium.

The amount of Lu complexed was determined to be 98% by the cation exchange method previously described.

Example 54

Preparation of 1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, yttrium(III) complex;

[Y(EA-D03A)]

The ligand, 1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (prepared by the procedure of Example 17) was converted to the mixed protonated-ammonium salt by chromatography on a strong cation exchange resin (SP-Sephadex™ C-25, Pharmacia). The column was in the protonated form and was washed with distilled water. A concentrated aqueous solution of the ligand was applied to the column, followed by washing the column with distilled water. The ligand was eluted from the column with 0.5 M NH 4 OH. The eluate was reduced to dryness on a rotary evaporator and dried in a vacuum oven.

A solution of Y(OAc)3.4H20 (0.203 g, 338.101 g/mol,

0.600 mmol) in 10 mL of distilled water was added to a hot solution of the mixed protonated ammonium salt ligand (0.300 g, 499.615 g/mol, 0.600 mmol) in 10 mL of distilled water. The solution was brought to reflux and the pH adjusted to about 7.0 with 0.1M NH 4 OH. After 15 min. reflux, the solution was cooled and reduced to dryness on a rotary evaporator. Excess ammonium acetate was removed by heating the white solid in an oil bath at about 105°C under vacuum. The title product (which contained about one equivalent of ammonium acetate) was obtained in a yield of 373 mg (99%) and was characterized by:

13C NMR (88°C, D2O, 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;

Fast atom bombardment mass spectrum, m/e 552 (positive ion, [M+H<+>]<+>), m/e 610 (negative ion, [M+0Ac~]”).

Example 55

Preparation of 1-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, samarium complex; [Sm(EA-D03A)].

When the procedure of Example 54 was repeated, using Sm(OAc)3.3H20 (0.229 g, 381.531 g/mol, 0.600 mmol) in place of Y(OAc)3.4H20, the title product (containing about one equivalent of ammonium acetate) was prepared in a yield of 408 mg (99%) and was characterized by:

<13>C NMR (88°C, D2O, 75 MHz)

23.8, 17.9, 51.4, 57.5, 58.1 (3C), 68.7, 73.4, 120.3, 132.2, 134.7, 185.0, 186.8 , 192.1;

Fast atom bombardment mass spectrum, m/e 615 (positive ion [M+H<+>]<+>, Sm isotopic pattern), m/e 673 (negative ion, [M+OAc~]~, Sm isotopic sample) .

Example 56

Preparation of 1-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, samarium(III) complex. 10 microliters of 1.48 mg/100 µl (0.03M) solution of l-[2-(4-nitrophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-tetraacetic acid (prepared by the method in Example 20) was added to 15 µl of SmCl3 solution (0.02M in 0.1N HCl) and added Sm-153. The volume was brought to 1 ml with NANOpure™ water. This solution was divided into two 500 µl fractions, and one fraction was heated for 1 hour at 100°C. Both heated and unheated samples were tested to determine the percent metal complexed using the cation exchange method described hereinbefore. The results showed 81% and 43% of the metal as complex for, respectively. the heated and unheated samples.

Example 57

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

The heated sample of Example 56 was divided into two aliquots. One aliquot was adjusted with HCl and the other with NaOH to give the pH values shown in the following table. After the desired pH had been achieved, the sample was allowed to stand for 10 min. and the amount of metal complexed was determined by the cation exchange method described earlier. The results are shown in the following table.

Example 58

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

Three µl of a 4.65 mg/100 ml solution of 1-[2-(4-aminophenyl)-ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (prepared by the procedure in Example 20 ) was added to a solution of 15 µl of SmCl3 (0.02M in 0.1N HCl) previously added to Sm-153. The volume was brought to 1 ml with NANOpure™ water. The pH of the solution was adjusted to 7.3 with NaOH. The solution was divided into two aliquots, and one aliquot was heated at 100°C for 1 hour.

Both heated and unheated samples were tested to determine the percent metal complexed by the cation exchange method described earlier. The results showed 96% and 93% of the metal as complex for, respectively. the heated and unheated samples.

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 samples in Example 58 were all divided into two aliquots. One aliquot of each sample was adjusted with HCl and the other with NaOH to give the pH values shown in the following table. After the desired pH had been achieved, the sample was allowed to stand for 10 min., and the amount of metal complexed was determined by the cation exchange method described earlier. The results are shown in the following table.

Example 60

Preparation of 1-(2-hydroxy-5-nitrobenzyl)-1,4,7,lo-tet razacycl ododecane-4,7,10-triacetic acid, samarium(III) complex.

A 3 x 10~<2>M solution of 1-(2-methoxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (prepared by the method in Example 25) was prepared by dissolving 3.9 mg in 260 pl of NANOpure™ water. A 10 µl aliquot of this solution was added to 15 µl of 2 x 10"<2>M SmCl3 (in H2O) spiked with 10 µl of Sm-153 in 0.1N HCl (3 x 10"<4>M). The volume was brought to one milliliter by adding 965 µl of DI H 2 O.

The pH was then brought to 7.0 with NaOH. The solution was split into two fractions of 500 µl each, and one fraction was heated for 1 hour at 100°C. Both heated and unheated samples were tested to determine the percent metal complexed by the previously described cation exchange method. The results showed 71% and 74% of the metal as complex for, respectively. the heated and unheated samples.

Example 61

pH stability of 1-(2-hydroxy-5-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, samarium(III) complex.

The heated and unheated sample from Example 60 were each split into two 250 µl aliquots. One aliquot was adjusted with HCl and the other with NaOH to give the pH values shown in the following table. After the desired pH had been achieved, the sample was allowed to stand for 10 min. and the amount of metal complexed was determined by the cation exchange method described earlier. The results are shown in the following table.

Example 62

Preparation of 1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, samarium(III) complex.

A 3 x 10~<2>M solution of 1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid (prepared by the method in Example 26) was prepared by dissolving 3.2 mg in 100 pl of NANOpure™ water. A 10 µl aliquot of this solution (diluted to 20 µl) was added to 15 µl of 2 x 10~<2>M SmCl 3 (in H 2 O) spiked with Sm-153. The volume was brought to 1 ml by adding 950 µl DI H 2 O and 20 µl 0.1N NaOH. This solution was split into two fractions of 500 µl each, and one fraction was heated for 1 hour at 100°C. Both heated and unheated samples were tested to determine the percent metal complexed by the cation exchange method described earlier. When less than 95% of the metal was complexed, the solution was passed through SP-Sephadex™ resin. The percentage of metal as complex was again determined. The results are shown in the following table.

Example 63

pH stability of 1-(2-hydroxy-5-aminobenzyl)-1,4,7 ,10-tetraazacyclododecane-4,7,10-triacetic acid, samarium(III) complex.

The heated and unheated purified chromatographed samples from Example 62 were each split into two 250 µl aliquots. One 250 µl aliquot was adjusted with HCl and the other with NaOH to give the pH values shown in the following table. When the desired pH had been achieved, the samples were allowed to stand for 10 min., and the percentage of metal as a complex was determined by the cation exchange method described earlier. The results are as shown in the following table:

Application methods - Biology

Bifunctional chelating agents that have within the same molecule a metal chelating group such as DTPA and a reactive linker (the arylamine) are known to be able to bind covalently to various targeted biomolecules that have specificity for cancer or tumor cell epitopes or antigens. Radionuclide complexes of such conjugates are useful in diagnostic and/or therapeutic applications as means of delivering the radionuclide to a cancer or tumor cell.

[Reference, Meares et al., Anal Biochem. 142. 68-78 (1984); see also discussion on pages 215-216 of J. Protein Chem. , 3.(2)

(1984) by Meares and Goodwin, more recent references are Meares, U.S. Patent 4,678,677, issued July 7, 1987, Warshawsky et al. U.S. patent 4,652,519, granted March 24. 1987 and Brechbiel et al., Inorg. chem., 2J5, 2772-2781 (1986). Understandably, by using radioactive or luminescent metals, the product can also be used for in vitro immunoassays.

Background information on

The applicability of the labeled antibodies depends on a number of factors, for example: 1) the specificity of the antibody, 2) the persistence or stability of the complex under the conditions of use (i.e. serum stability), and 3) the integrity of the antibody, i.e. that the specificity of the antibody and immunoreactivity is not affected by the labeling process.

The stability or persistence of the complex is of utmost importance to the effectiveness of the radionuclide antibody conjugate as a diagnostic and/or therapeutic agent. Kinetic lability can lead to instability, e.g. in serum, and dissociation of the radionuclide from the complex. It thus reduces the diagnostic and therapeutic efficiency. Moreover, it represents a greater potential for general radiation damage to normal tissue. [Cole, et al., J. Nucl. Down.,

28, 83-90 (1987)].

Binding of radionuclide to antibody

Binding of radionuclide to antibody can be accomplished by either binding BFC to the antibody using methods well known in the art, followed by chelation of the radionuclide under conditions compatible with the antibody, or alternatively, conjugation of the antibody to the formed (ambient or elevated temperature) metal - BFC complex.

[Meares et al., Acc.Chem.Res. 17, 202-209 (1984)]. Examples are provided.

Example ZA (Comparative)

Conjugation of 1-(4-isothiocyanatebenzyl)-diethylenetriaminepentaacetic acid, samarium-153 complex with IgG and F(ab')2 in CC-49;[15<3>Sm(SCN-Bz-DTPA)]-IgG and [15 <3>Sm(SCN-Bz-DTPA)]-F(ab')2 fragment.

1-(4-isothiocyanatebenzyl)diethylenetriaminepentaacetic acid, samarium-153 complex [<153>Sm(SCN-Bz-DTPA)] was prepared by

mix 150 µl <153>Sm in 0.1N HCl with 9 µl of 1-(4-isothiocyanate-benzyl)diethylenetriaminepentaacetic acid to which HEPES buffer (0.5M, pH 8.9, about 30 µl) had been added to bring pH to about 6. To conjugate, 22.5 x 10"<9> mol IgG or F(ab')2 in CC-49 (about 1 x 10~<4>M concentration of protein in 50 mmol HEPES , pH 8.5) was mixed with the 153Sm complex, and the pH was adjusted to 8.9 by adding a sodium carbonate solution (1.0M, 12-15 µl)• The conjugation was carried out at room temperature (about 25°C) for about 3 hours The <153>Sm complex labeled IgG or F(ab')2 was isolated and characterized in the same manner as described in Example XII.

Experimental for biology

Examples I & II and Comparative Examples A-D IN VIVO

SCREENING OF BIFUNCTIONAL CHELATES.

The stability of certain rare earth chelates has been investigated by in vivo testing in animals. E.g. report Rosoff, et al. in the International Journal and Applied Radiation and Isotopes 14, 129-135 (1963) on the distribution of radioactive rare earth chelates in mice, in relation to certain amino-carboxylic acids. It was found that in vivo "it is the competition between the chelating agent and the body's constituents (inorganic and organic) for the rare earth ions that determines their deposition and excretion11. The strong earth ions of this invention are believed to dissociate very little and be excreted, while the weak chelates and medium-strength chelates known in the art dissociate more easily, and are thus deposited in organs such as the liver. However, the concentration of radionuclide in the liver is not always due to weak complex formation, but in some cases is due to the affinity that the metal chelate has for the liver. Chelates in fact, have been prepared and used for evaluation of liver function [Fritzberg, Alan R., Radiopharmaceuticals: Progress and Clinical Perspectives, Vol. 1, 1986; U.S. Patents 4,088,747 and 4,091,088].

The biodistribution of the yttrium and samarium chelates of the compound in Example 3, an example of a strong rare earth chelate, was determined, and percent dose to the liver was used as an in vivo screening method to qualitatively assess the stability of the chelates. Chelates of NTA and EDTA are included for comparison. Ogå's samarium was injected as samarium chloride in unchelated form as a control.

Sprague-Dawley rats weighing 150 to 200 g were purchased from Charles River Laboratories. These animals were housed in cages and given water and food ad libitum. The animals were acclimatized for at least five days before use. Before injection of complex, the animals were placed under a heat lamp (15 to 30 min.) to dilate the tail vein. The animals were then restrained, the tail cleaned with alcohol, and the animal injected (50 to 200 µl) via the tail vein. After the injection, the animal was placed in another cage for 2 hours, after which the animal was euthanized by throat slitting. The animal was then dissected, the parts rinsed with deionized water, dried and weighed in a tared counting glass. At least three standards of the same material as injected were prepared and counted with the animal tissues. Percentage dose is the number of counts in the organ divided by the number of counts in the standard multiplied by 100 (see the following table).

<*>The complexes were prepared at a ligand/metal ratio of 1:1 for Examples I and II; in 5:1 for example A; and at about 300:1 for examples B and C.

Examples III and E.

The 1:1 complexes of yttrium (spiked with a trace amount of <90>Y) with the ligand of Example 3, which is BA-DOTA, and EDTA (comparative E) were prepared by methods described previously. Several 100 µl aliquots were then transferred to separate centrifuge tubes. Excess of Y(III) was added, so that the total volume change is minimized and the time is noted. 1/2 hour after the metal addition, the complex percentage was determined by the cation exchange method described earlier, and this was compared with the original complex quantity. Percent complex in relation to added metal gives one indication of the lability of the ligand-metal complex. The results are given in

the table.

Example IV

Preparation of 1-[2-4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, samarium-153 complex; [<153>Sm(SCN-EA-D03A)] complex.

To 100 µl of 153Sm in 0.1N HCl was added 5 µl of SCN-EA-D03A (5 mM in 50 mM HEPES, pH 8.2) (prepared in Example 18). This was mixed in a vortex mixer, and HEPES buffer (0.5M,

pH 8.9) was added gradually (about 25 µl total) to adjust the pH to about 7. The degree of chelation was monitored by HPLC on a GF-250 column (HPLC system I). Yields around 50% were achieved.

Example V

Preparation of c-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex; [<153>Sm(PA-DOTA)] complex.

To 150 microliters (µl) of <153>Sm solution in 0.1N HCl (about 4.6 mCi) was added 1 µl sodium acetate (2.OM) and 9 µl of α-[2-(4-aminophenyl)ethyl ]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mixed potassium ammonium salt (5 mmol in 50 mmol HEPES, pH 8.5, prepared by the method of Example 9). The mixture was mechanically shaken and titrated gradually with HEPES buffer (0.5M, pH 8.9; about 31 µl added) to pH 7. This was then heated at 98°C in a sand bath for 1 hour. After completion, 5 µl of the mixture was used for analysis on a Mono-Q™ column on HPLC system II, eluted with a gradient solvent system (0-15 min, from 0 to 100% B; where A = water, and B = 1.OM ammonium acetate and 0.1 mmol EDTA). Yields of 85-95% based on 15<3>Sm were obtained. α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the samarium-153 complex thus prepared was characterized by comparison with the identical non-radioactive the sample, by their respective chromatographic behavior on Mono-Q™ and GF-250 columns. Further evidence of the presence of the radioactive complex was determined by conversion to the isothiocyanate derivative and its subsequent conjugation to antibody. The complex has also been prepared without heating (at ambient temperature) by incubation for 6 to 18 hours, resulting in 70-80% yield.

Example VI

Preparation of c-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex; [15<3>Sm(SCN-PA-DOTA)] complex.

To the reaction mixture obtained in Example V was added

2 µl of HEPES buffer (0.5M, pH 8.9), 2 µl thiophosgene and 0.2 ml chloroform. The mixture was mechanically vigorously shaken 2 to 3 times for a few seconds every hour. The chloroform layer was discarded, and the aqueous layer containing essentially the desired product was retained and further purified. The yield of α-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium 153 complex was 85-90%, based on <153 >Sm activity measurement by HPLC on GF-250 column using HPLC system I. For purification, the aqueous layer was passed through a Sep-Pak™ C-18 cartridge, and eluted with 90% acetonitrile in water. The first 300 µl of effluent was discarded, and the SCN derivative emerging in the next 900 µl was characterized by HPLC on GF-250. The recovery of the <153>Sm activity was better than 90%. Most of the solvent was then evaporated over a period of 1.5 to 2 hours, and the residue was used for conjugation to antibody.

Example VII

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

To 200 µl of <153>Sm solution in 0.1N HCl was added 1 µl of sodium acetate (2.OM) and 12 µl of α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4, 7,10-tetraacetic acid (5 mmol in 50 mmol HEPES, pH 8.5) (prepared by the method in example 3). The mixture was mechanically shaken and titrated gradually with a HEPES buffer (0.5M, pH 8.9; about 38 µl added) to pH 7. This was then heated at 98°C in a sand bath for 1 hour. After completion, 5 µl of the mixture was used for analysis on a Mono-Q™ column (HPLC system II, eluted with a gradient solvent system: 0-15 min., from 0 to 100% B; where A = water, and B = 1.0 M ammonium acetate and 0.1 mmol EDTA). Typically, yields of 85-95% based on <153>Sm were obtained. The complex has also been prepared by incubating the mixture at room temperature for 12-18 hours, which gave a yield of the title product of 80-90%. α-(4-Aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the samarium 153 complex thus prepared was characterized by comparison with an authentic sample by their chromatographic behavior on MonoQ ™ column, conversion to the isothiocyanate derivative and its subsequent conjugation to antibody.

Example VIII

Preparation of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex; [<153>Sm(SCN-BA-DOTA)] complex.

To the reaction mixture obtained in Example VII, 2 µl of HEPES buffer (0.5 M, pH 8.9), 2 µl of thiophosgene and 0.2 ml of chloroform were added. It was vigorously mechanically shaken 2 or 3 times in a couple of seconds every hour. The chloroform layer was discarded, and the aqueous layer containing essentially the desired product was collected and further purified. The yield of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the samarium-153 complex, as analyzed by HPLC on a GF-250 column based on <153 > The Sm activity using HPLC system I was over 90%. For purification, the aqueous layer was passed through a Sep-Pak™ C-18 cartridge, and eluted with 90% acetonitrile in water. The first 0.3 mL of effluent was discarded, and the desired product emerged in the next 1.2 mL, with 86-93% recovery. Most of the solvent was then evaporated over a period of about 2 hours, and the residue was used for conjugation to antibody.

Example IX

Conjugation of 1-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,lo-tet raazacyclododecane-4,7,10-triacetic acid, samarium-153 complex to antibody; [15<3>Sm(SCN-EA-D03A)]-IgG conjugate.

The antibody used was CC-49, a murine monoclonal IgG that binds to an epitope of TAG-72, a tumor-associated antigen. To conjugate, 187 µl of the antibody solution (1.20 x 10"<4>M in 50 mM HEPES, pH 8.5) was mixed with 4.5 x 10~<8> mol of 15<3>Sm(SCN- EA-D03A) prepared as described in Example IV, followed by the addition of a sodium carbonate solution (1.0M) to raise the pH to 8.9. The reaction was allowed to proceed for 2 hours at room temperature. Upon completion, <153>Sm labeled IgG isolated by centrifugal gel filtration on Sephadex G-25 (2.2 ml) disposable columns, and further purified by HPLC on a GF-250 column, eluted with citrate buffer (0.25M, pH 7.4). The fractions containing the labeled IgG were pooled , concentrated and exchanged (3x) into PBS using Centricon™ concentrators. The specific activity of <153>Sm labeled IgG prepared in this way was about 0.16 pCi/pg protein. The integrity of [<153>Sm (EA-D03A)]-IgG preparation was verified by HPLC (Sivakoff, S.I., BioChrom. 1(1), 42-48 (1986))] and standard biochemical methods, e.g., sodium dodecyl sulfate:polyacrylamide gel electrophoresis and autoradiography, and solid-phase radioimmunoassay (RIA). [See David Colcher et al., Cancer Res. 43, 736-742 (1983)].

Example X

Conjugation of l-[2-(4-isothiocyanatephenyl)ethyl]-l,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, samarium-153 complex with fragment F(ab')2 in CC-49 ;[15<3>Sm(SCN-EA-D03A)] fragment F(ab')2 in CC-49.

The F(ab')2 fragment in CC-49, prepared by enzymatic cleavage according to the method described by Lamoyi and Nisonoff [E. Lamoyi and A Nisonoff, J. Immunol. Methods, 56, 235-243

(1983)], (225 µl of 1 X 10~<4>M in 50 mM HEPES, pH 8.5) was mixed with 2.9 x 10~<8> mol of <153>Sm(SCN-EA- D03A) prepared as described in Example IV. Sodium carbonate (1.0M, about 5 µl) was added to bring the pH to 8.9, and the reaction was continued for about 2.5 hours. Upon completion, the <153>Sm-labeled fragment was isolated, and characterized as described in Example IX.

Example XI (A and B)

In vivo localization of [153Sm(EA-D03A)]-IgG and [15<3>Sm(EA-D03A)]-F(ab')2.

The utility of the 15<3>Sm(EA-D03A) labeled IgG (from Example IX) and F(ab')2 in CC-49 (from Example X) was demonstrated by the uptake of the labeled substances by human tumor xenografts in athymic mouse. Thus, athymic (Nu/Nu, CD1 background) female mice were inoculated subcutaneously (S.C.) (0.1 ml/well) with the human colon carcinoma cell line, LS-174 T (about 1 x 10<6> cells/animal). About 2 weeks after inoculation, each animal was injected via the tail vein with about 2 pCi (15-30 pg) of <153>Sm labeled antibody in PBS. The mice were euthanized after different time intervals. After bleeding, the tumor and selected tissue were excised and weighed, and the radioactivity was measured in a gamma counter. Counts per my. (CPM) for <153>Sm in each tissue was determined and expressed as CPM per grams of tissue per injected dose multiplied by 100 (% injected dose/gram). The results are shown in figures 1-14 and tables IA and Ib. 15<3>Sm(SCN-Bz-DTPA) labeled IgG and F(ab')2 in CC-49 (from Example ZA) were included in the study for comparison. The results are shown in Tables IC and ID).

Example XII

Conjugation of α-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex to antibody; [<153>Sm(SCN-PA-DOTA)]-IgG conjugate.

The antibody used was CC-49, a murine monoclonal IgG that binds to an epitope of TAG-72, a tumor-associated antigen. To conjugate, 178 µl of the antibody solution (1.26 x 10"<4> M in 50 mmol HEPES, pH 8.5) was mixed with 3.4 x 10"<8 >mol of α-[2-(4- isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium complex (prepared as described in Example VI), followed by the addition of a sodium carbonate solution (1.0M, about 17 µl to raise the pH to about 8.9. The reaction was allowed to proceed for 2 hours at room temperature. After completion, <153>Sm-labeled IgG was isolated by centrifugal gel filtration on Sephadex™ G-25 (2.2 mL) disposable columns, and further purified by HPLC on GF-250 column, eluted with citrate buffer (0.25M, pH 7.4).The fractions containing the labeled IgG were pooled, concentrated and exchanged (3 times) into PBS using Centricon™ concentrators. The specific activity of <153>Sm-labeled IgG prepared in this manner was about 0.16 pci/pg protein.The homogeneity and integrity of α-[2-(4-isothiocyanatephenyl)-ethyl]-1,4,7 ,10-tetraazacyclododecane-1, The 4,7,10-tetraacetic acid, samarium-153 complex IgG preparation was verified by HPLC and standard biochemical procedures as in Example IX.

The following example is an alternative to making labeled antibody conjugates, which first involves conjugation of BFC to the antibody, and subsequent chelation to give the radionuclide BFC-labeled Ab.

Example XIIA

Preparation of α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid Ab CC49 conjugate (IgG C<C>49-PA-DOTA) ; and subsequent chelation with <153>Sm to form <153>Sm-labeled antibody (IgG CC49-PA-DOTA-153Sm).

The <153>Sm(PA-DOTA) labeled antibody can be prepared by first coupling BFC, e.g. α-[2-4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (SCN-PA-DOTA), to an antibody at pH 8-9, followed by chelation with <153>Sm at pH 6 at room temperature for several hours.

In a typical experiment, IgG CC-49 was concentrated and exchanged three times into a carbonate buffer (50 mM, pH 9.1) in a Centricon™ concentrator (molecular weight cutoff of 30 K) to leave a solution with antibody concentration greater than 1.5

x 10~<4>M. To form the conjugate, 161 µl of the antibody solution, containing 25 x 10<9> mol of IgG CC-49 was mixed with 5 µl of SCN-PA-DOTA (5 mM concentration in the same carbonate buffer, prepared by the method of Example 18). The mixture was allowed to stand at room temperature for 5 hours, and ended by filtration through the 30 K membrane in the Centricon™ concentrator at 5000 rpm in the Sorvall RT centrifuge. The antibody conjugate was further washed with 2 ml of a 0.25 M DPA solution in PBS at pH 7.4 and five times (2 ml each time) with MES buffer (20 mM, pH 5.8); centrifuged for 30 min. after each wash.

Finally, the PA-DOTA-IgG conjugate was concentrated to a minimum volume (about 100 µl) and its integrity checked by HPLC analysis on a GF-250 column. To the purified conjugate was added a mixture of <153>Sm in 0.1N HCl (50 µl) and 20 µl of an MES buffer (1.0M, pH 6), mixed in a vortex mixer, and allowed to stand room temperature overnight. After termination by centrifugal gel filtration, the introduced amount of <153>Sm, estimated by HPLC analysis, was 0.22 BFC-Sm/antibody. That <153>Sm was associated with the antibody by chelation with BFC, which was bound covalently to the antibody, and not because non-specific binding, was demonstrated by comparison with results from the control experiment. In the control experiment, IgG CC-49 solution was mixed with <153>Sm mixture under identical conditions. The antibody similarly isolated had no appreciable amount of <153>Sm associated with it. This therefore showed that non-specific binding of Sm-153 to the antibody did not occur under these conditions.

Example XIII

Conjugation of α-[2-(4-isothiocyanatephenyl)-ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex to fragment F(ab')2 in CC -49; [153Sm(SCN-PA-DOTA)]-F(ab')2 fragment.

The F(ab')2 fragment in CC-49 [prepared by enzymatic cleavage according to the method described by (E. Lamoyi et al., J. Immunol. Methods 56, 235-243 (1983)], (225 µl of 1 x 10"<4>M in 50 mmol HEPES, pH 8.5) was mixed with 2.9 x 10~<8> mol of oc-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10 -tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex prepared as described in Example VI Sodium carbonate (1.0M, about 9 µl) was added to bring the pH to about 8.9, and the reaction was continued for about 2.5 hours.<153>Sm-labeled fragment was isolated and characterized as described in Example IX.The specific activity was about 0.4 pCi/pg.

Example XIVfA oa Bl

In vivo localization of the conjugate with α-[2-(4-isothiocyanatephenyl )ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex labeled IgG and F(ab 2; [153Sm(SCN-PA-DOTA]-IgG and [<1>5<3>Sm(SCN-PA-DOTA)]-F(ab')2•

The utility of the conjugate of α-[2-(4-isothiocyanatephenyl)-ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex labeled IgG and F(ab' )2 with CC-49 (from examples XII and XIII) was demonstrated by uptake of the labeled substances by human tumor xenografts in athymic mice. The biolocalization was determined using the method described in Example XI. Results are shown in Figures 1 to 7 for IgG and Figures 8 to 14 for F(ab')2, also Tables IIA and IIB.

The conjugate of 15<3>Sm(SCN-Bz-DTPA) with IgG and F(ab')2-labeled substances (prepared from Example ZA) was included in the study for comparison. (See tables IC and ID.)

Example XV

Conjugation of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex with antibody [<153>Sm(SCN-BA-DOTA)]- IgG.

Whole IgG from CC-49 (174 µl of 1.2 x 10"<4>M in 0.25M HEPES, pH 8.7) was mixed with 2.0 x 10~<8> mol of c-(4-isothiocyanatephenyl ) - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 (2.8 mCi) prepared according to Example VIII, followed by the addition of a sodium carbonate solution (1.0M, about 2 pl) to maintain the pH at about 8.7. The reaction was allowed to proceed for 2.5 h at room temperature. After completion, <153>Sm labeled IgG was isolated by centrifugal gel filtration on Sephadex™ G-25 (2.2 ml) disposable columns , and further purified by HPLC on a GF-250 column, eluted with citrate buffer (0.025M, pH 7.4). The fractions containing the labeled IgG were pooled, concentrated and exchanged (3 times) into PBS using Centricon ™ concentrators Homogeneity and integrity of the samarium-153 labeled IgG preparation was verified by HPLC and standard biochemical procedures as described in Example IX.

Example XVI

Conjugation of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex with fragment F(ab')2 in CC-49; [<153>Sm(SCN-BA-DOTA)] fragment F(ab')2

in CC-49.

F(ab')2 in CC-49 (84 pl of 2.4 X 10"<4>M in 0.25M HEPES

buffer, pH 8.7), prepared by enzymatic cleavage according to the method described by Lamoyi et al. was mixed with 2.1 x 10~28 mol of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex (prepared by the method in example VIII). Sodium carbonate (1.0M, about 2 µl) was added to bring the pH to 8.7, and the reaction was continued for about 2 hours. At completion, the <153>Sm labeled fragment was isolated and characterized as described in Example IX.

Example XVII (A and B)

In vivo localization of α-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex labeled IgG (Example XV) and F(ab')2 (Example XVI); [<153>Sm(BA-DOTA)]-IgG and [<153>Sm(BA-DOTA]-F(ab')2.

The in vivo study was carried out according to the protocol described in Example XI, and the results are shown in figures 1 to 14 and

tables HIA and UIB.

Example XVIII (A and B)

In vivo localization of c-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 177Lu complex labeled IgG and F(ab')2; [<177>Lu(PA-DOTA)]-IgG and [<177>Lu(PA-DOTA)]-F(ab')2.

The title compounds were prepared and in vivo studies were performed according to the protocol described in Examples V, VI, XI, XII

and XIII and the results are shown in Tables IVA and IVB.

Example XIX (A and B)

In vivo localization of c-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, yttrium-90 complex labeled IgG and F(ab')2 ; [<90>Y(PA-DOTA)]-IgG and [90Y(PA-DOTA)]-F(ab')2.

The title compounds were prepared and in vivo studies were performed according to the protocol described in Example XI, with the exception that the tissues were dissected and counted by liquid scintillation and the results are shown in Tables VA and VB.

Example XX

In vitro pH stability of [<153>Sm(BFC)]-CC-49-IgG and [<177>LU(BFC)]-CC-49-IgG.

[<153>Sm(BFC)]-CC-49-)IgG or [177Lu(BFC)]-CC-49-IgG was allowed to stand in a 0.2M NaOAc buffer at pH 6.0, 4.0 and 2, 8 at room temperature, at a protein concentration of about 5 x 10~<6>M with about 0.5 complex/antibody. Samples were withdrawn at specific time intervals and analyzed by HPLC (GF-250 column) for dissociation of 153Sm or 177Lu activity from the protein. Usually the study was carried out for five days or until 90% of the radioisotopes had been dissociated. The results are expressed as the initial rate of radioisotope loss per day and night. The results show the superior stability of <153>Sm(PA-DOTA), <177>Lu(PA-DOTA, <153>Sm(BA-DOTA), 153Sm(PA-DOTMA) and 153Sm(MeO-BA-DOTA) , compared to the standard [<153>Sm(Bz-DTPA)] complex at acidic pH, the results are shown in the table below.

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

Example XXI (A and B)

In vivo localization of ot-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, samarium-153 complex labeled IgG and F(ab</>) 2; [<153>Sm(MeO-BA-DOTA)]-IgG and [<153>Sm(MeO-BA-DOTA)]-F(ab')2.

The title compounds were prepared according to the methods described in Examples V, VI, XII and XIII, and the in vivo studies were carried out by the methods of Example XI. The results are shown in tables VIA and VIB.

Example XXII

Preparation of 15<3>Sm(PA-DOTMA) complex.

To 100 microliters (pl) of <153>Sm (0.3 x 10-3M in 0.01N HCl;

5 mCi) was added 6 µl of PA-DOTMA (high Rf diastereomer prepared by the method of Example 29; 5 mM in MilliQ™ water), 20 µl of a MES buffer (1.0M, pH 6) and 1 µl of a HEPES buffer (0.5 M, pH 8.7). This solution was mixed on a vortex mixer, and the final pH of the mixture was about 6. After heating the mixture at 90°C for 30 min., it was analyzed by HPLC on a GF-250 column for degree of complexation, yielding 90% or better was usually achieved. Characterization of the <153>Sm-PA-DOTMA complex was performed by comparison with the non-radioactive-Sm complex, which had been synthesized and characterized independently as described in Example 42

Example XXIII

Preparation of 15<3>Sm(SCN-PA-DOTMA).

To the <153>SmPA-DOTMA complex prepared in Example XXII and which had been cooled for 20 min. at room temperature, 10 µl of a 1% thiophosgene solution in 90% acetonitrile-water medium was added. The mixture was vigorously mixed on a vortex mixer, giving

stand at room temperature for between 5 and 20 min. The reaction took place immediately as followed by HPLC analysis. To purify the activated <153>Sm complex, it was extracted 3 times (200 µl each) with chloroform and the aqueous layer was passed through a PRP cartridge (PRP cartridge = Mini-clean™ cartridge from Alltech Associates, Deerfield , IL) pretreated with 5 ml of methanol and 5 ml of an MES buffer (20 mM, pH 5.8).. After washing with 5 ml of the MES buffer and 5 ml of Milli-Q™ water, it was extracted with 900 µl of 90% acetonitrile to recover about 90% of the 153Sm activity, with the first 100 µl discarded. The mixture was evaporated to dryness under reduced pressure at temperatures below 40°C, and the residue containing most of the title product was used for conjugation with the antibody.

Example XXIV fA oa B )

Conjugation of <153>Sm(SCN-PA-DOTMA) with IgG and F(ab')2 CC49.

Typically, the antibody was concentrated and exchanged into a carbonate buffer (pH 9.1 or 9.5, 50 mM) in a Centricon™ concentrator (molecular weight cutoff of 3OK) to result in a protein concentration of 1.5 x 10~<4>M or higher. To conjugate, a small volume of the carbonate buffer, that required to bring the final protein concentration to 1.5 x 10~<4>M, is added to the isothiocyanate derivative of 153Sm(PA-DOTMA) (prepared in Example XXIII), followed by of the concentrated antibody, equimolar with the BFC-<153>Sm complex. It was mixed on a vortex mixer and allowed to react at room temperature for 1 hour or until 40 to 50% of a <153>Sm was bound to the antibody, as shown by HPLC analysis on a GF-250 column.

The labeled antibody was isolated by two consecutive centrifugal gel filtrations on Sephadex<T>2M G-25 columns

(2.2 ml). The homogeneity, integrity and immunoreactivity of the labeled antibody was analyzed by HPLC analysis and standard biochemical techniques described previously.

Example XXV (A and B)

Biodistribution studies of <153>Sm(PA-DOTMA) labeled IgG and F(ab')2 CC49.

The studies were carried out in a similar manner as described in Example XI. The results are shown in tables VHA and VIIB. See Figures 15-28.

Example XXVI

Preparation of 177Lu (PA-DOTMA) complex.

To 30 µl of 17<7>Lu (6 x 10"<3>M in 1.1N HCl; 4 mCi) was added 36 µl of PA-DOTMA (prepared by the procedure in Example 29); 5 mM solution in milli- Q™ water).The solution was mixed on a vortex mixer, and 115 µl of an MES buffer (1.0M, pH 6.0) was added to neutralize the solution to about pH 5.5 to 6. This was heated at 90°C for 30 min to result in better than 90% chelation.The mixture was passed through a PRP cartridge pretreated with 5 ml of methanol and 5 ml of an MES buffer (20 mM, pH 5.8). It was washed with 5 ml of milli-Q™ water.The complex extracted in 1000 µl of 90% acetonitrile was responsible for 78% of initial <177>Lu activity (discarding the first 100 µl of the extract).

Example XXVII

Preparation of 17<7>Lu(SCN-PA-DOTMA).

To the <177>Lu (PA-DOTMA) complex in 90% acetonitrile obtained

in Example XXVI, 6 µl of a 10% thiophosgene in 90% was added

acetonitrile. This solution was mixed on a vortex mixer, and after 20 min. analyzed for formation of the isothiocyanate derivative by HPLC. The turnover is usually quantitative. Removal of the solvent and excess thiophosgene was achieved by evaporation under reduced pressure at temperatures below 40°C for 1 to 2 hours. The residue was used for conjugation with the antibody.

Example XXVIII

Conjugation of 17<7>Lu(PA-DOTMA) to IgG CC49.

The antibody IgG CC49 in carbonate buffer (50 mM, pH 9.1) was mixed with an equimolar amount of the isothiocyanate derivative (prepared by the method of Example XXVII) in the same buffer. The reaction was continued for 70 min. at an antibody concentration of 1.5 x 10"<4>M, and the labeled antibody was isolated and characterized according to the methods described in Example IX.

Example XXIX

Long-term biodistribution studies of <177>Lu(PA-DOTMA) and <177>Lu(PA-DOTA) labeled IgG CC49.

The long-term animal test was performed with <177>Lu labeled antibody, taking advantage of its long half-life (161 hours). It was performed with Balb/c mice over a three week period, according to protocols described in Example XI. The results are shown in Table VIIIA. See figures 29-34.

In the figures plotting data from the following tables, the symbols used were:

In the accompanying figures, the given data shall represent the following:

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention described herein. It is intended that the description and examples be considered solely as examples, with the true scope and spirit of the invention indicated by the following claims.

Claims (5)

1. Diagnostically applicable compound characterized by the formula where M is a cation selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y and Sc, each Q is independently hydrogen or (CH<5>)pCO 2 R; Q<1> is hydrogen or (CH<5>)wC02<R>; each R is independently hydrogen, benzyl or C 1 -C 4 alkyl; provided that at least two of the sum of Q and Q<1> must be different from hydrogen, and that when Q<1> is hydrogen, R<3> must be different from hydrogen; each R<5 > is independently hydrogen or C 1 -C 6 alkyl; X and Y are both independently hydrogen or can together with a neighboring X and Y form an additional carbon-carbon bond; n is 0 or 1; m is an integer from 0 to 10; p is 1 or 2; r is 0 or 1; w is 0 or 1; with the proviso that n is only 1 when X and/or Y form an additional carbon-carbon bond, and the sum of r and w is 0 or 1; R 2 is hydrogen, nitro, amino. isothiocyanate, semicarbazide, thiosemicarbazide, maleic imide, bromoacetamide or carboxyl; R<3 > is C-L-C 6 alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen; R<4> is hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, maleic imide, bromoacetamide or carboxyl; with the proviso that R2 and R<4> cannot both be hydrogen, but one of R2 and R<4> must be hydrogen; or a physiologically acceptable salt thereof. 2. Connection according to claim 1, characterized in that it is the 1-[2-(isothiocyanate-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid metal complex, The 1-(2-methoxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid metal complex, The 1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid metal complex, a-[2-(4-amino-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic acid 4,7,10-tris-(R)-methylacetic acid, 1- the isopropyl-4,7,10-trimethylester metal complex, α-[2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic acid 4,7,10-tris-(R)-methylacetic acid metal complex, The α-[2-(4-isothiocyanate-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic acid-4,7,10-tris-(R)-methyl-acetic acid metal complex , The α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid metal complex. The α-(4-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,10-triacetic acid metal complex, The α-(4-amino-phenyl)-1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid metal complex, or-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid metal complex, α-[2-(4-nitro-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the 1,4,7,10-tetramethyl ester metal complex, cx-[2-(4-amino-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetramethyl ester metal complex, α-[2-(4-amino-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid metal complex, α-[ 2-(4-isothiocyanatephenyl)ethyl ]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid metal complex, The α-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetic acid metal complex, α-(2-Methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the tetra-ammonium salt metal complex, or α-(2-Methoxy-5-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, the tetraammonium metal salt complex. 3. Compound characterized by the formula wherein each Q is independently hydrogen or (CH<5>)pCO 2 R; Q<1> is hydrogen or (CH<5>)wC02<R>; each R is independently hydrogen, benzyl or C 1 -C 4 alkyl; provided that at least two of the sum of Q and Q<1> must be different from hydrogen, and that when Q<1> is hydrogen, R<3> must be different from hydrogen; each R<5> independently is hydrogen or C1-C4 alkyl; X and Y are both independently hydrogen or can together with a neighboring X and Y form an additional carbon-carbon bond; n is 0 or 1; m is an integer from 0 to 10; p is 1 or 2; r is 0 or 1; w is 0 or 1; with the proviso that n is only 1 when X and/or Y form one additional carbon-carbon bond, and the sum of r and w is 0 or 1; R 2 is hydrogen, nitro, amino. isothiocyanate, semicarbazide, thiosemicarbazide, maleic imide, bromoacetamide or carboxyl; R 3 is C 1 -C 1 -Alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen; R 4 is hydrogen, nitro, amino, isothiocyanate, semicarbazide, thiosemicarbazide, maleic imide, bromoacetamide or carboxyl; with the proviso that R2 and R4 cannot both be hydrogen, but one of R<2> and R<4> must be hydrogen; or a physiologically acceptable salt thereof, 4. Connection, characterized in that it is 1-[2-(isothiocyanate-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, 1-(2-Methoxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, 1-(2-hydroxy-5-aminobenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid, cx- [2-(4-amino-phenyl) ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic acid 4,7,10-tris-(R)-methylacetic acid, 1- isopropyl 4,7,10-trimethyl ester, oe- [2-(4-aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic acid 4,7,10-tris-(R)-methylacetic acid, a;- [ 2-(4-isothiocyanate-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic acid-4,7,10-tris-(R)-methylacetic acid, α-(4-amino-phenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, α;-(4-amino-phenyl)-1,4,7,10-tetraazacyclododecane- l, 4,10-triacetic acid, cx-(4-amino-phenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid of-(4-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, a-[2-(4-nitro-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetramethyl ester, α-[2-(4-amino-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetramethyl ester, ot-[2-(4-amino-phenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetic acid, oe-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Qf-(2-methoxy-5-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetic acid, ot-(2-methoxy-5-aminophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, tetraammonium salt, or or-(2-Methoxy-5-isothiocyanatephenyl)-1,4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid, tetraammonium salt. 5. Connection, characterized in that it is the reaction product of an antibody or antibody fragment reacted with oe-[2-(4-isothiocyanatephenyl)ethyl]-1,4,7,10-tetraazacyclododecane-1-(R,S)-acetic acid- 4,7,10-tri(R-methylacetic acid) complex.