WO1993012112A1

WO1993012112A1 - Bicyclopolyazamacrocyclocarboxylic acid complexes, their conjugates processes for their preparation, and use as radiopharmaceuticals

Info

Publication number: WO1993012112A1
Application number: PCT/US1992/010663
Authority: WO
Inventors: Garry E. Kiefer; Jaime Simon
Original assignee: The Dow Chemical Company
Priority date: 1991-12-10
Filing date: 1992-12-10
Publication date: 1993-06-24
Also published as: EP0575583A4; ZA929578B; FI933508A; EP0575583A1; FI933508A0; CA2103553A1; HU9302308D0; AU3275293A; MX9207165A; KR930703331A; IL104062A0

Abstract

Complexes of bicyclopolyazamacrocyclocarboxylic acid compounds with a metal ion, e.g. ?153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 99mTc, 67Ga, 68Ga, 105Rh, 97Ru, 111In, 113mIn or 115m¿In ion, are disclosed. The complexes can be covalently attached to a biologically active molecule, e.g. an antibody or antibody fragment, to form conjugates. The complexes and conjugates are useful as radiopharmaceutical agents for therapy and/or diagnostic purposes. Processes to prepare the complex and conjugate are discussed.

Description

BICYCLOPOLYAZAMACROCYCLOCARBOXYLIC ACID COMPLEXES, THEIR CONJUGATES, PROCESSES FOR THEIR PREPARATION, AND USE AS RADIOPHARMACEUTICALS

This invention concerns complexes that contain as the ligand bicyclopolyazamacrocyclocarboxylic acids complexed with metal ions, and comjugates thereof, for use as radiopharmaceuticals, especially for the treatment and/or diagnosis of cancer. The processes for their preparation are also provided.

The delivery of radionuclides to different organ and tissue targets has been the objective of many research efforts for both diagnostic and therapeutic purposes. Various molecules have been tried that would carry the active component to the desired site and yet be stable at least until the site has been reached by the delivery system. Halogenated (e g ¹³¹ l and ^88mBr) organic molecules have been used. Thus iodinated hippuran has been used to study renal function, e.g. J. Nucl. Med. 23, 377-380 (1982). Also labelling a monoclonal antibody with ¹³¹l has been proposed for the detection and therapy of cancer, e.g. Cancer Res. 44, 5744-5751 (1984).

Metalic radionuclides offer a variety of nuclear properties and chemistries Thus, for example, ²⁰¹Tl, ⁶⁷Cu, ^99mTc, ⁹⁰Y and various isotopes of In and Ga are only a few examples of radioisotopes that have been used for disgnostic imaging and/or therpy. Of these metals, the chemistry of ^99mTc has been explored the most for use as a radiopharmaceutical . For example, Tc-diphosphonates are used to image the skeletal system [see Subramanian et al., Radiology 149, 823-828 (1983)]. Loberg etal. [J. Nucl. Med. 16, 533 (1975)] were able to study liver function with lipophilic ^99mTc complexes in which the Tc existed in a + 3 oxidation state and the overall charge of the iminodiacetic acid complex was -1. Deutsch etal. [Science 213, 85 (1981)] was able to prepare Tc complexes with As and P containing ligands that localized in the heart. These compounds contained a Tc(lll) core with an overall charge of the complex of + 1 Also Volkert et al. [Int'l. J Appl. Rad. Isotopes 35, 467-470 (1974)] were successful in delivering Tc(lll) to brain tissue

However, since ^99mTc is a pure gamma emmiter , it is limited only to diagnostion applications. Therefore, there has been a need for particle emmiting radioisotope complexes and/or conjugates which would be useful in therapy. Deutsch etal. [Corina Int'l. , Veronai and Raven Press, pp.733-740 (1990)] have used the combination of ¹⁸⁶Re and a diphosphonate to treat bone tumors. Also Simon et al. (U.S. Patent 4,898,724) teach the use of ¹⁵³Sm and other rare earth radionuclides ϊn combination with aminophosphonic acids for the treatment of bone pain and tumors.

The development of bone metastases is a common and often catastrophic event for a cancer patient. The pain, pathological fractures, frequent neurological deficits and forced immobility caused by these metastatic lesions significantly decrease the quality of life for the cancer patient. The number of patients that contract metastatic disease is large since nearly 50% of all patients who contract breast, lung or prostate carcinoma will eventually develop bone metastases. Bone metastases are also seen in patients with carcinoma of the kidney, thyroid, bladder, cervix and other tumors, but collectively, these represent less than 20% of patients who develop bone metastases. Metastatic bone cancer is rarely life threatening and occasionally patients live for years following the discovery of the bone lesions. Initially, treatment goals center on relieving pain, thus reducing requirements for narcotic medication and increasing ambulation. Clearly, it is hoped that some of the cancers can be cured.

The use of radionuclides for treatment of cancer metastaticto the bone dates back to the early 1950's. It has been proposed to inject a radioactive particle-emitting nuclide in a suitable form for the treatment of calcific lesions. It is desirable that such nuclides be concentrated in the area of the bone lesion with minimal amounts reaching the softtissue and normal bone. Radioactive phosphorus (P-32 and P-33) compounds have been proposed, but the nuclear and biolocafization properties limit the use of these compounds. (E. Kaplan, etal., J. Nucl. Med 1(1), 1, (I960); U.S. Patent 3,965,254).

Another attempt to treat bone cancer has been made using phosphorus compounds containing a boron residue. The compounds were injected into the body

(intravenously) and accumulated in the skeletal system. The treatment area was then irradiated with neutrons in order to activate the boron and give a therapeutic radiation dose (U.S. Patent 4,399,817).

The use of Re-186 complexed with a diphosphonate has also been proposed.

[L Mathϊeu et al.. Int. J. Applied Rad. & Isotopes, 30, 725-727 (1979); J. Weinenger, A. R.

Ketring et al., J. Nucl. Med., 24(5), P125 (1983)]. However, the preparation and purification needed forthis complex limits its utility and wide application.

Stron tϊum-89 has also been proposed for patients with metastatic bone lesions. However, the long half-life (50.4days), high blood levels and low lesion to normal bone ratios limit the utility. [N. Firusian, P. Mellin, C. G. Schmidt, J Urology, 116, 764 (1976); C G Schmidt, N Firusian, Int. J. Clin. Pharmacol., 93, 199-205, (1974)].

A palliative treatment of bone metastases has been reported wh^;ch employed 1-131 labelled α-amino-(3-iodo-4-hydroxybenzylidene)diphosphonate [M. Eisenhut, J Nucl Med., 25(12), 1356-1361 (1984)]. The use of radioiodine as a therapeutic radionudide is less than desirable due to the well known tendency of iodine to localize i n the thyroid. Eisenhut lists iodide as one of the possible metabolites of this compound.

The use of radionuclides for calific tumor therapy or relief of bone pain is discussed in published European patent application 176,288, where the use of Sm-153, Gd- 159, Ho-166, Lu-177 or Yb-175 complexed with a ligand such as ethylenediaminetetraacetic acid (EDTA) or hydroxyethylenediaminetriacetic acid (HEEDTA) is disclosed. A macrocyclic system having a 1,4,7,10-tetraazacyclododecane moiety complexed with Sm-153, Gd-159, Ho-166, Lu-177 or Yb-175 for calific tumor therapy or relief of bone pain is discussed in U.S. Patent 5,059,412 which complex is very stable and has a lower charge than the complex disclosed in published European patent application 176,288.

Functionalized chelants, or bifunctional coordinators, are known to be capable of being covalently attached to an antibody having specif icity for cancer or tumor cell epitopes or antigens. Radionudide complexes of such antibody/chelant conjugates are useful in diagnostic and/ortherapeutic applications as a means of conveying the radionuclidetoa cancer or tumor cell. See, for example, Meares et al., Anal. Biochem. 142, 68-78, (1984), and Krejcarek et al., Biochem. and Biophys. Res. Comm. 77, 581-585 (1977).

Aminocarboxylic acid chelating agents have been known and studied for many years. Typical of the aminocarboxylic acids are nitrilotriacetic acid (NTA), ethyl- enediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), di- ethylenetriaminepentaacetic acid (DTPA), trans-1 ,2-diamino cyclohexanetetraacetic acid (CDTA) and 1, 4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) Numerous bifunctional chelating agents based on aminocarboxylic acids have been proposed and prepared. For example the cyclic dianhydride of DTPA [Hnatowich etal. Science 220, 613-615, (1983); U.S. Patent 4,479,930] and mixed carboxycarbonic 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 reported. When the anhydrides are coupled to proteins the coupling proceeds via formation of an amide bond thus leaving four of the original five carboxymethyl groups on the diethylenetriamine (DETA) backbone [Hnatowich et al. Int. J. Appl. Isot 33, 327- 332, (1982)]. In addition, U.S. Patents 4,432,907 and 4,352,751 disclose bifunctional chelating agents useful for binding metal ions to "organic species such as organic target molecules or antibodies." As in the above, coupling is achieved via an amide group through the utilization of 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 discloses several complex salts formed from the anion of a complexing acid for use in various diagnostic techniques. Conjugation via a carboxyl group of the complexing acid is taught which gives a linkage through an amide bond. Inthe J. Radioanal. Chem. 57(12), 553-564 (1980), Paiketal. disclose the use of p-nitrobenzylbromide in a reaction with a "blocked" diethylenetriamine, i.e. bis-(2- phthalimidoethyl)amϊne followed by deblocking procedures and carboxymethylation using chloroaceticacid, to give N'-p-nitrobenzyldiethylenetriamine N,N,N",N"-tetraaceticacid. Again, since the attachment is through a nitrogen, a tetraacetic acid derivative is obtained. Conjugation of the bifunctional chelating agent and chelation with indium is discussed. Substitution on the nitrogen atom is also taught by Eckelman, et al. in the J. Pharm. Sci. 64(4), 704-706 (1975) by reacting amines such as "ethylenediamine or diethylenetriamine with the appropriate alkyl bromide before carboxymethylation." The compounds are proposed as . potential radiopharmaceutical imaging agents.

Another class of bifunctional chelating agents based on aminocarboxylic acid functionality is also well documented in the literature. Thus, Sundberg, Meares, et al. in the J. Med. Chem. 17(12), 1304 (1974), disclosed bifunctional analogs of EDTA. Representative of these compounds are 1-(p-aminophenyl)-ethylenediaminetetraaceticacid and 1-(p- benzenediazonium)ethylenediaminetetraaceticacid. Coupling to proteins through the para- substϊtuent and the binding of radioactive metal ions to the chelating group is discussed. The compounds are also disclosed in Biochem. andBiophys. Res. Comm.75(1), 149 (1977), and in U.S. Patents 3,994,966 and 4,043,998. It is important to note thatthe attachment of the aromatic group to the EDTAstructure is through a carbon of the ethylenediamine backbone. Optically active bifunctional chelating agents based on EDTA, HEDTA and DTPA are disclosed in U.S.4,622,420. In these compounds an alkylene group links the aromatic group (which contains the functionality needed for attachment to the protein) to the carbon of the polyamine which contains the chelating functionality. Other references to such compounds include Brechbiel etal., 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 disclosed in U S 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 through a ring carbon of the cyclic polyamine backbone. Thus, a linker, attached atone end to a ring carbon of the cyclic polyamine, is also attached at its other end to a functional group capable of reacting with the protein.

Another class of bifunctional chelating agents, also worthy of note, consists of compounds wherein the chelating moiety, i.e. the aminocarboxylic acid, of the molecule is attached through a nitrogen to the functional group of the molecule containing the moiety capable of reacting with the protein. As an example, Mikola et al. in patent application (WO 84/03698, published 9/27/1984) disclose a bifunctional chelating agent prepared by reacting p- nitrobenzyl bromide with DETA followed by reaction with bromoacetic acid to make the aminocarboxylic acid. The nitro groupis reduced to the corresponding amine group and is then converted to the isothiocyanate group by reaction with thiophosgene. These compounds are bifunctional chelating agents capable of chelating lanthanides which can be conjugated to bio-organic molecules for use as diagnostic agents. Since attachment of the linker portion of the molecule is through one of the nitrogens of the aminocarboxylic acid, then one potential aminocarboxyl group is lost for chelation. Thus, a DETA-based bifunctional chelant containing four (not five) acid groups is prepared. In this respect, this class of bifunctional chelant is similar to those where attachment to the protein is through an amide group with subsequent loss of a carboxyl chelating group.

Recently Carney, Rogers, and Johnson disclosed (3rd. Int'l. Conf. on Monoclonal

Antibodies For Cancer: San Diego, California - 2/4-6/88) abstracts entitled "Absence of

Intrinsically Higher Tissue Uptake from lndium-11 1 Labeled Antibodies: Co-administration of lndium-111 and lodine-125 Labeled B72.3 in a Nude Mouse Model" and "Influence of Chelator Denticity on the Biodistribution of lndium-1 1 1 Labeled B72.3 Immunoconjugates in Nude Mice". The biodistribution of indium-111 complexed with an EDTA and DTPA bifunctional chelating agent is disclosed. Attachment of the aromatic ring to the EDTA/DTPA moieties is through an acetate methylene. Also at a recent meeting D.K. Johnson et al. [Florida Conf. on Chem. in Biotechnology, April 26-29 (1988), Palm Coast, FL] disclosed bifunctional derivatives of EDTA and DTPA where a p-isothiocyanatobenzyl moiety is attached at the methylene carbon of one of the carboxymethyl groups. Previously Hunt et al. in U.S. Patents 4,088,747 and 4,091 ,088 (1978) disclosed ethylenediaminediacetic acid (EDDA) based chelating agents wherein attachment of an aromatic ring to the EDDA moiety isthrough the alkylene or acetate methylene. The compounds are taught to be useful as chelates for studying hepatobiliary function. The preferred metal is technetium-99m. lndium-111 and indium-1 13m are also taught as useful radionuclides for imaging.

Such uses of other complexes are known using radio frequency to induce hyperthermia (Japanese Kokai Tokkyo Koho JP 61 , 158,931) and fluorescent-lmmunoguided therapy (FIGS) [K. Pettersson et al., Clinical Chem. 29(1), 60-64 (1983) and C. Meares et al., Acc. Chem. Res. 17 , 202-209 (1984)].

Consequently, it would be advantageous to provide a complex that does not readily dissociate, that exhibits rapid whole body clearance except from the desired tissue, and conjugates with an antibody to produce the desired results.

Advantageously, the present invention provides a new type of a stable metal complex, especially with metals that are rare earths or pseudo-rare earths in their chemistry. This invention teaches the use of these complexes and that the variance of their charge and lipophilic character may favorably alter their biodistribution when introduced into an animal. The conjugates of these complexes with a biologically active material, such as an antibody, are also a part of this invention. These complexes and conjugates may be formulated with suitable pharmaceutical carriers and administered to a mammal for disgnosis and/or therapy.

. The present invention is directed to novel complexes comprising a ligand that is a bicydopolyazamacrocyclocarboxylicacid of the formula

where: X and Y are independently H, OH, C₁-C₃ alkyl or COOH;

R⁷ is H or OH; and

R⁴ is H, NO₂, NH₂, isothiocyanato, semicarbazido,thiosemicarbazido, maleimido,

bromoacetamido or carboxyl;

with the proviso that at least two R terms must be

A = CH, N, C-Br, C-CI, C-OR¹, C-OR², N⁺-R³ X-, or

R¹ = H, C₁-C₅ alkyl, benzyl, or benzyl substituted with at least one R⁴;

R² is C₁-C₁₆ alkylamino;

R³ is C₁- C₁₆ alkyl, benzyl, or benzyl substituted with at least one R⁴;

R⁴ is defined as before;

X is Cl -, Br-, l- or H₃CCO₂-;

Q and Z independently are CH, N, N⁺-R³ X-, C-CH₂-OR¹ or C-C(O)-R⁵;

R³ is defined as above;

R⁵ is -O-(C₁-C₃ alkyl), OH or NHR⁶;

R⁶ is C₁- C₅ alkyl or a biologically active material;

X^'is defined as above; and

with the proviso that:

a) when Q, A or Z is N or N ⁺-R³X-, then the other two groups must be CH;

b) when A is C-Br, C-CI, C-OR¹ or C-OR², then both Q and Z must be CH;

c) thesum of the R², R⁴and R⁶terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R⁵ and when one of Q or Z is C-C(O)-R⁵, then A must be CH; and

complexed with a metal ion of ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re,

¹⁴²Pr, ^99mTc, ⁶⁷Ga, ⁶⁸Ga, ¹⁰⁵Rh, ⁹⁷Ru, ¹¹¹ln, ^113mln or ^115mln; or

pharmaceutically-acceptabie salts thereof.

Bifunctional complexes of Formula (I) are desirable to prepare the conjugates of this invention. Such ligands must have:

one Rterm is

where R⁴ and R⁷ are defined as above; or A is C-OR¹, C-OR², where R¹ and R² are defined as above or

where R⁴ is defined as above; or

A is CH, and one of Q or Z is CH and the other is C-C(O)-R⁵ or C-CH₂- OR¹, where R¹ and R⁵ are defined as above;

especially those ligands where R⁵ is NHR⁶, where R⁶ is a biologically active material.

The complexes of Formula (I) use various metal ions, usually in the +3 state, selected from: samarium (¹⁵³Sm), lutetium (¹⁷⁷Lu), holmium (¹⁶⁶Ho), yttrium (⁹⁰Y), scandium (⁴⁷Sc), rhenium (¹⁸⁶Re) or (¹⁸⁸Re), praseodymium (¹⁴²Pr),technetium (^99mTc), gallium (⁶⁷ Ga) or (⁶⁸ Ga), or indium (¹¹¹ln) or (^115mIn); with ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁴²Pr, 9^9mTc, ⁶⁷ Ga, ⁶⁸Ga, ^{11 1}ln, ^113mln or ^115mln being preferred; with ¹⁵³Sm, ¹⁷⁷Lu, ¹⁶⁶Ho, ⁹⁰Y, ^99mTc, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹In, ^113mln or ^115mln being especially preferred; and with ¹⁵³Sm, ¹⁷⁷Lu or ¹⁶⁶Ho being most preferred. Complexes having gamma emissions, such as ^99mTc, ⁶⁸Ga, ⁶⁷ Ga, ¹¹¹ln, ^113mln, or ⁹⁷Ru, are useful as diagnostic agents. Complexes having particle emissions, such as ¹⁴⁹Pm, ¹⁴²Pr,⁹⁰Y, or ¹⁷⁵Yb, are useful as therapeutic agents. Complexes having both gamma and particle emissions, such as ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁶⁶Ho, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁰⁵Rh, or ^115mln, are useful as both diagnostic and therapeutic agents. The complexes so formed can be used by themselves or can be attached, by being covalently bonded, to an antibody or fragment thereof and used for diagnostic or therapeutic purposes. Such conjugates and complexes are useful as diagnostic and/ortherapeutic agents.

The complexes and conjugates of this invention can be modified to provide a specific overall charge. For example, when the metal ion is + 3 the following can be obtained:

(A) an overall neutral charge - when

Ris

and X and Y are all equal to H;

(B) an overall + 1 charge -when

one of A, Q or Z is N ⁺ -R³ X-, where R³ and X- are defined as above; and the three R terms are

Both the complexes and conjugates may be formulated to be in a pharmaceutically acceptable form for administration to an animal.

Use of the complexes or conjugates of this invention for diagnosis or therapy of disease states such as cancer is possible.

Use ofthe ligands of this invention with other metal ions for diagnosis of disease states such as cancer is possible. The use of those complexes and conjugates is discussed in another copending application.

The complex has the ligand of Formula (I) numbered for nomenclature purposes as follows:

The present invention concerns development of radiopharmaceutical agents having synthetic modifications to the chelate enabling site specific delivery of the

radiopharmaceutical agent to a desired tissue. The advantage being increased delivery of the radiopharmaceutical agent in the areas of interest based upon tissue affinity. The specificity of the complex of Formula (I) may be controlled by adjusting the total charge and lipophilic character ofthe complex. The overall range of the charge of the complex is from -3 to + 1. For example, for a complex having 2 or more PO₃H₂ groups, the overall charge is highly negative and bone uptake is expected; whereas when the overall charge of the complex is 0 (thus neutral), the com pi ex may have the ability to cross the blood brain barrier and normal brain uptake may be possible.

Tissue specificity may also be realized by ionic or covalent attachment of the chelate to a naturally occuring or synthetic molecule having specificity for a desired target tissue. One possible application of this approach is through the use of chelate conjugated monoclonal antibodies which would transport the radioactive chelate to diseased tissue enabling diagnosis and therapy. Additionally, the present radiopharmaceutical agents of Formula (I) which are neutral in charge are particularly preferred for forming the conjugates of this invention since undersirable-ionic interactions between the chelate and protein are minimized which preserves the antibody immunoreactivity.

While notwϊshing to be bound by theory, it is believed thatwhen a charged complex of the invention is made (e.g. + 1 for heart), the variations in that chelate ionic charge can influence biolocalization. Thus, if the antibody or other directing moiety is also specific for the same site, then the conjugate displays two portions to aid in site specific delivery.

The terms used in Formula (I) and forthis invention are further defined as follows. "C₁-C₃ alkyl", "C₁-C₅ alkyl", "C₁-C₁₈ alkyl", include both straight and branched chain alkyl groups. An "animal" includes a warmblooded mammal, preferably a human being.

"Biologically active material" refers to, for example, a dextran, peptide, or molecules that have specific affinity for a receptor, or preferably antibodies or antibody fragments.

"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 portion of an antibody having specificity toward a desired epitope orepϊtopes. When using the term "radioactive metal chelate/antibody conjugate" or "conjugate", the "antibody" is meant to include whole antibodies and/or antibody fragments, including semisynthetic or genetically engineered variants thereof.

Possible antibodies are 1116-NS-19-9 (anti-colorectal carcinoma), 1116-NS-3d (anti-CEA), 703D4 (anti-human lung cancer), 704A1 (anti-human lung cancer), CC49 (anti-TAG-72), CC83 (anti- TAG-72) and B72.3. The hybrϊdoma cell lines 1116-NS-19-9, 1116-NS-3d, 703D4, 704A1, CC49, CC83 and B72.3 are deposited with the American Type Culture Collection, having the accession numbers ATCC HB 8059, ATCC CRL 8019, ATCC HB 8301 , ATCC HB 8302, ATCC HB 9459, ATCC HB 9453 and ATCC HB 8108, respectively.

As used herein, "complex" refers to a complex ofthe compound ofthe invention, e.g. Formula (I), complexed with a metal ion, where at least one metal atom is chelated or sequestered; "conjugate" refers to a metal ion chelate that is covalently attached to an antibodyor antibody fragment. The terms "bifunctional coordinator", "bifunctional chelating agent" and "functionalized chelant" are used interchangeably and refer to com pounds that have a chelant moiety capable of chelating a metal ion and a moiety covalently bonded to the chelant moiety that is capable of serving as a means to covalently attach to an antibody or antibody fragment.

The bifunctional chelating agents described herein (represented by Formula I) can be used to chelate or sequester the metal ions so as to form metal ion chelates (also referred to herein as "complexes"). The complexes, because ofthe presence ofthe functionalizing moiety (represented by R², R⁴ or R⁶ in Formula I), can be covalently attached to biologically active materials, such as dextran, molecules that have specific affinity for a receptor, or preferably covalently attached to antibodies or antibody fragments. Thus the complexes described herein may be covalently attached to an antibody or antibody fragment or have specific affinity for a receptor and are referred to herein as "conjugates".

As used herein, "pharmaceutically-acceptable salt" means any salt or mixtures of salts of a complex or conjugate of formula (I) which is sufficiently non-toxic to be useful in therapy or diagnosis of animals, preferrably mammals. Thus, the salts are useful in accordance with this invention. Representative of those salts formed by standard reactions from both organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, glutamic, gluconic, d- camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic and cinnamic acids and other suitable acids. Also included are salts formed by standard reactions from 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 complexes or conjugates of formula (I) where the salt is potassium, sodium or ammonium. Also included are mixtures of the above salts.

The complexes or conjugates of the present invention contain a ligand of Formula (I). The ligands are prepared by various processes. Typical general synthetic approaches to such processes are provided by the reaction schemes given below

In Scheme 1 , the compounds of Formula (I) are prepared wherein Q, A and Z =

CH, and all three R =

X

|

-C-CO₂H

|

Y

Scheme 2 prepares the compounds of Formula (I) wherein A = C-Br, and Q and Z = CH.

Scheme 3 prepares the compounds of Formula (I) wherein A =

R⁴ = H, NO₂, NH₂ or SCN; and Q and Z = CH.

Scheme 4 prepares the compounds of Formula (I) wherein A = C-OR², where R² = C₁- C₅ alkylamino; and

Q and Z = CH.

Scheme 5 prepares the compounds of Formula (I) wherein A = CH; and one of Q or Z = CH and the other Q or Z = C-C(O)-R⁶ or C-CH₂-R⁶, where R⁶ is defined as before.

Scheme 6 prepares the compounds of Formula (I) wherein Z = C-CH₂-OBz or C- C(O)-R⁵where R⁵ = -O-(C₁-C₃ alkyl), OH or NHR⁶, where is defined as before; and

Qand A = CH.

Scheme 7 prepares the compounds of Formula (I) wherein A = N or N ⁺-R⁵X-; R⁵ = C₁-C₁₆ alkyl and is X- halide; and

Q and Z = CH.

Scheme 8 prepares the compounds of Formula (I) wherein Q = N⁺-R⁵X-, where R⁵ = C₁-C₁₆ alkyl and X- = halide; and

Aand Z = CH.

Scheme 9 prepares the compounds of Formula (I) wherein Q = N or N + -R⁵ X-, where R⁵ = C₁-C₁₆ alkyl and

X- = halide; and

Aand Z = CH.

Scheme 10 preparesthe compounds of Formula (I) wherein Rterm at the 6 position is

where R⁴ = NO₂ or NH₂; and

A. Q and Z = CH.

Scheme 11 prepares the compounds of Formula (I) wherein the Rterm at the 9 position is

where R⁴ = NO₂ or NH₂; and

A,Q and Z = CH.

In the above Schemes, the general process discription illustrates specific steps that may be used to accomplish a desired reaction step. The general description of these process steps follows.

The synthetic Scheme 1 begins with a halogeπation of commercially available bis- pyridyl alcohol (1) using thionyl chloride. Similar procedures for converting an alcohol to an electrophilic substrate, such as treatment with toluenesulfonyl chloride, HBr or HCI, should also result in a similarily reactive product which would work well in subsequent ring closure reactions. Macrocyclization procedures are numerous in the literature and the desired tetraazamacrocycle (3) was prepared according to the method of Stetter et al., Tetrahedron 37, 767-772 (1981). More general procedures have since been published which give good yields of similar macrocycies using milder conditions [A. D. Sherry etal., J. Org. Chem. 54, 2990-2992 (1989)]. Detosylation ofthe intermediate macrocycle [(3) to yield (4)] was accomplished under acidic conditions in good yield. Reductive detosylation procedures are also well known in the literature and can be adapted to the present reaction sequence.

Schemes 10 and 11 delinate a synthetic approach which introduces an aromatic nitrobenzyl substitutentatone ofthe macrocyclic nitrogen positions. Typically, the macrocyclic amine is mono-N-functioπalized in an organic solvent such as acetoπitrile or DMF at room temperature using a non-nudeophiltc base such as potassium carbonate. Additional functionalizatron ofthe remaining nitrogen positions is then preformed by methods and conditions described in previous Schemes. After the introduction ofthe desired chelating moieties, the πitro group isreduced using platinum oxide and hydrogen in water. In this form, the chelating agent is compatible with conjugation techniques which will enable attachment to larger synthetic or natural molecules.

The metal ions used to form the complexes of this invention are ¹⁵³Sm, ¹⁷⁷Lu, 1⁵⁹Gd, ¹⁴⁹Pm, ^{14 0}La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ^99mTc, ⁶⁷ Ga, ⁶⁸ Ga, ¹⁰⁵Rh, ⁹⁷Ru, ¹¹¹ ln, ^113mln or ^115mln. The anion present is halide, preferrably chloride, or salt free (metal oxide).

The complexes are prepared by methods well known in the art. Thus, for example, see Chelating Agents and Metal Chelates, Dwyer & Mellor, Academic Press (1964), Chapter 7. See also methods for making amino acids in Synthetic Production and Utilization of Ami no Acids, (edited byKameko,etaI.) John Wiley & Sons (1974). An example ofthe preparation of a complex involves reacting a bϊcyclopolyazamacrocyclocarboxylicacid with the metal ion under aqueous conditions at a pH from 5 to 7. The complex formed is by a chemical bond and results in a stable radionudide composition, e.g. stable to the disassociation ofthe radionudide from the ligand.

The complexes ofthe present invention are administered at a ligand to metal molar ratio of at least about 1:1, preferably from 1 :1 to 3:1, more preferably from 1 : 1 to 1.5: 1. A large excess of ligand is undesirable since uncomplexed ligand may be toxic to the animal or may result in cardiac arrest or hypocalcemic convulsions.

The antibodies or antibody fragments which may be used in the conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art, see for example, Kohler and Milstein [Nature, 256, 495-497 (1975); and fur. J. Immunol., 6, 51 1-519 (1976)]. Such antibodies normally have a highly specific reactivity. In the antibody targeted conjugates, antibodies directed against any desired antigen or hapten may be used. Preferably the antibodies which are used in the conjugates are monoclonal antibodies, or fragments thereof having high specificity for a desired epitope(s). Antibodies used in the present invention may be directed against, for example, tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules. Some examples of antibodies or antibody fragraments are 1116-NS-19-9, 11 16-NS-3d, 703D4, 704A1 , CC49, CC83 and B72.3. All of these antibodies have been deposited in ATCC. A more complete list of antigens can be found in U.S. Patent 4, 193,983. The conjugates of the present invention are particularly preferred for the diagnosis of various cancers.

This invention is used with a physiologically acceptable carrier, excipient or vehicle therefor. The methods for preparing such formulations are well known. The formulations may be in the form of a suspension, injectable solution or other suitable formulations. Physiologically acceptable suspending media, with or without adjuvants, may be used.

An "effective amount" ofthe formulation is used for diagnosis. The dose will vary depending on the disease and physical parameters ofthe animal, such as weight. In vivo diagnostics are also contemplated using formulations of this invention.

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

Claims

WHAT IS CLAIMED IS:

1. Acomplexwhich comprises a bicyclopolyazamacrocyclocarboxylic acid compound of the formula

wherein:

R is

where:

X and Y are independently H, OH, C₁-C₃ alkyl or COOH;

R⁷ is H or OH; and

R⁴ is H, NO₂, NH₂, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;

with the proviso that at least two R terms must be

A = CH, N, C-Br, C-CI, C-OR¹, C-OR², N ⁺-R³ X-, or

where: R¹ = H, C₁-C₅ alkyl, benzyl, or benzyl substituted with atleast one R⁴;

R²is C₁-C₁₆alkylamino;

R³ is C₁-C₁₆ alkyl, benzyl, or benzyl substituted with at least one R⁴;

R⁴ is defined as before;

X- is Cl-, Br-, |- or H₃CCO₂-;

Q and Z independently are CH, N, N ⁺-R³ X-, C-CH₂-OR¹ or C-C(O)-R⁵;

R³ is defined as above;

R^s is-O-(C₁- C₃ alkyl), OH or NHR⁶;

R⁶is C₁-C₅ alkyl or a biologically active material;

X- is defined as above; and

with the proviso that:

a) when Q, A or Z is N or N ⁺-R³ X-, then the other two groups must be CH;

b) when A is C-Br, C-Cl, C-OR¹ or C-OR², then both Q and Z must be CH;

c) the sum ofthe R², R⁴and R⁶ terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R⁵ and when one of Q or Z is C-C(O)-R⁵, then A must be CH;

¹⁴²Pr, ^99mTc, ⁶⁷ Ga, ⁶⁸Ga, ¹⁰⁵Rh, ⁹⁷Ru, ¹¹¹ln, ^113mlnor ^115mln; or

pharmaceutically-acceptable salts thereof.

2. A complex of Claim 1 wherein the metal ion is ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,

¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho,⁹⁰Y, ⁴⁷Sc, ¹⁴²Pr, ^99mTc, ⁶⁷ Ga, ⁶⁸Ga, ¹¹¹ In, ^{11 3m}ln or ^115mln.

3. A complex of Claim 2 wherein the metal ion is ¹⁵³Sm, ¹⁷⁷Lu, ¹⁶⁶Ho, ⁹⁰Y, ^99mTc, 6⁷Ga, ⁶⁸ Ga, ^{11 1}In, ^113mIn or ^{11 5m}ln.

4. A complex of Claim 3 wherein the metal ion is ¹⁵³Sm, ¹⁷⁷Lu or ¹⁶⁶Ho.

5. A complex of Claim 1 wherein X and Y are H.

6. A complex of Claim 1 wherein A, Q and Z are CH.

7. A complex of Claim 1 wherein Q, A and Z are CH; and one R term is

where: X, Y, R² and R⁴ are defined as in Claim 1.

8. A complex of Claim 1 wherein A is C-OR¹, C-OR², where R¹ and R² are defined as in Claim 1 or

where R⁴ is defined as in Claim 1.

9. A complex of Claim 1 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-R⁵ or C-CH₂-OR¹, where R¹ and R⁵ are defined as in Claim 1.

10. A complex of Claim 9 wherein R⁵ is NHR⁶, where R⁶ is a biologically active material.

11. A conjugate comprising a bicyclopolyazamacrocyclocarboxylic acid compound ofthe formula

wherein:

R is

where:

X and Y are independently H, OH, C₁-C₃ alkyl or COOH;

R⁷ is H or OH; and

R⁴ is H, NO₂, NH₂, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido,

bromoacetamido or carboxyl;

with the proviso that at two R terms are

A = CH, N, C-Br, C-CI, C-OR', C-OR², N ⁺-R³ X-, or

R¹ = H, C₁-C₅ alkyl, benzyl, or benzyl substituted with at least one R⁴;

R² is C₁- C₁₆ alkylamino;

R⁴ is defined as before;

χ-is Cl-, Br-, r- or H₃CCO₂- ;

Q and Z independently are CH, N, N ⁺-R³ X-, C-CH₂-OR¹ or C-C(O)-R⁵;

R³ is defined as above;

R^s is -O-(C₁-C₃ alkyl), OH or NHR⁶;

R⁶ is C₁-C₅ alkyl ora biologically active material;

X- is defined as above; and

with the proviso that:

a) when Q, A or Z is N or N ⁺-R³X-, then the other two groups must be CH;

b) when A is C-Br, C-CI, C-OR¹ or C-OR², then both Q and Z must be CH;

c) the sum ofthe R², R⁴and R⁶ terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R^s and when one of Q or Z is C-C(O)-R⁵, then A must be CH;

¹⁴²Pr,^99mTc, ⁶⁷ Ga, ⁶⁸ Ga, ¹⁰⁵Rh, ⁹⁷Ru, ¹¹¹ln, ^{11 3m}ln or ^{11 5m}ln; and

covalently attached to a biologically active material; or

pharmaceuticaily-acceptable salts thereof.

12. A conjugate of Claim 11 wherein the metal ion is ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁴²Pr,^99mTc, ⁶⁷ Ga, ⁶⁸Ga, ^{11 1}In, ^113mln or ^{11 5m}ln.

13. A conjugate of Claim 11 wherein the biologically active material is a dextran, a polypeptide, a molecule that has specific affinity for a receptor, or an antibody or antibodyfragment.

14. Aconjugate of Claim 13 wherein the antibody or antibody fragment is a monoclonal antibody or fragment thereof.

15. A conjugate of Claim 14 wherein the antibody or antibody fragment is B72.3.

16. A conjugate of any one of Claims 12-15 wherein the metal ion is ¹⁵³Sm, 1⁷⁷Lu, ¹⁶⁶Ho, ⁹⁰Y, ^99mTc, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹ In, ^113mln or ^{11 5m}ln.

17. A conjugate of Claim 16 wherein the metal ion is ¹⁵³Sm, ¹⁷⁷Lu or ¹⁶⁶Ho.

18. A conjugate of Claim 11 wherein X and Y are H.

19. A conjugate of Claim 11 wherein A, Q and Z are CH.

20. A conjugate of Claim 1 1 wherein Q, A and Z are CH; and one R term is

where: X and R⁴ are defined as in Claim 11.

21. A conjugate of Claim 1 1 wherein Q, A and Z are CH; and one Rterm is

where: R⁴and R⁷ are defined as in Claim 11.

22. A conjugate of Claim 11 wherein A is C-OR¹, C-OR², where R¹ and R² are defined as in Claim 1 1 , or

where R⁴ is defined as in Claim 11.

23. A conjugate of Claim 11 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-R⁶, where R⁶ is defined as in Claim 11.

24. A conjugate of Claim 23 wherein R⁶ is NHR⁷, where R⁷ is a biologically active material

25. A pharmaceutical formulation comprising a complex as claimed in any one of Claims 1-10 with a pharmaceuti cally-acceptable carrier.

26. A pharmaceutical formulation comprising a conjugate as claimed in any one of Claims 1 1-24 with a pharmaceutically-acceptable carrier.

27. A method forthe diagnosis of a disease state in an animal which comprises administering to said animal an effective amount of the formulation of Claim 25.

28. A method forthe diagnosis of a disease state in an animal which comprises administering to said animal an effective amount ofthe formulation of Claim 26.

29. The complex as claimed in any one of Claims 1-10for use asa pharmaceutϊcal.

30. The conjugate as claimed in any one of Claims 11-24 for use as a pharmaceutical.

31. A kit for use as a therapeutic agent which has as one component a ligand of Formula (I) as claimed in Claim 1, and as a second component a metal ion as claimed in

Claim 1.

32. A process for preparing the complex as claimed in any one of Claims 1-10, which comprises reacting a bicydopolyazamacrocydocarboxylic acid compound as claimed in Claim 1 with a metal ion of ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ^99mTc, ⁶⁷Ga, ⁶⁸ Ga, ¹⁰⁵Rh, ⁹⁷Ru, ¹¹¹ In, ^113mln or ^{11 5m}ln under aqueous conditions at a pH from 5 to 7.