EP0696290A1 - Acides bicyclopolyazamacrocyclophosphoniques, leurs complexes et conjugues, destines a etre utilises en tant qu'agents de contraste, et leurs procedes de preparation - Google Patents

Acides bicyclopolyazamacrocyclophosphoniques, leurs complexes et conjugues, destines a etre utilises en tant qu'agents de contraste, et leurs procedes de preparation

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EP0696290A1
EP0696290A1 EP93911136A EP93911136A EP0696290A1 EP 0696290 A1 EP0696290 A1 EP 0696290A1 EP 93911136 A EP93911136 A EP 93911136A EP 93911136 A EP93911136 A EP 93911136A EP 0696290 A1 EP0696290 A1 EP 0696290A1
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compound
alkyl
terms
term
cooh
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Garry E. Kiefer
Jaime Simon
Joseph R. Garlich
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Dow Chemical Co
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Dow Chemical Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
    • A61K49/106—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being cyclic, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • This invention concerns l igands that are bicyclopolyazamacrocyclophosphonic acids, and complexes and conjugates thereof, for use as contrast agents in magnetic resonance imaging (MRI). Some ligands and complexes are also useful as oral care agents and as scale inhibiting agents in water treatment systems.
  • MRI magnetic resonance imaging
  • Some ligands and complexes are also useful as oral care agents and as scale inhibiting agents in water treatment systems.
  • Background MRI is a non-invasive diagnostic technique which produces well resolved cross-sectional images of soft tissue within an animal body, preferably a human body. This technique is based upon the property of certain atomic nuclei (e.g. water protons) which possess a magnetic moment [as defined by mathematical equations; see G. M.
  • the relaxation time consists of two parameters known as spin-lattice (T1) and spin-spin (T2) relaxation and it is these relaxation measurements which give information on the degree of molecular organization and interaction of protons with the surrounding environment.
  • paramagnetic chelates possessing a symmetric electronic ground state can dramatically affect the T1 and T2 relaxation rates of juxtaposed water protons and that the effectiveness of the chelate in this regard is related, in part, to the number of unpaired electrons producing the magnetic moment [Magnetic Resonance Annual , 231-266, Raven Press, NY (1985)]. It has also been shown that when a paramagnetic chelate of this type is administered to a living animal, its effect on the T1 and T2 of various tissues can be directly observed in the magnetic resonance (MR) images with increased contrast being observed in the areas of chelate localization.
  • MR magnetic resonance
  • paramagnetic metal ions there are a number of paramagnetic metal ions which can be considered when undertaking the design of an MRI contrast agent.
  • the most useful paramagnetic metal ions are gadolinium (Gd +3 ), iron (Fe +3 ), manganese (Mn +2 ) and (Mn + 3 ), and chromium (Cr +3 ), because these ions exert the greatest effect on water protons by virtue of their large magnetic moments.
  • GdCl 3 non-complexed form
  • these metal ions are toxic to an animal, thereby precluding their use in the simple salt form. Therefore, a fundamental role of the organic chelating agent (also referred to as a ligand) is to render the paramagnetic metal non-toxic to the animal while preserving its desirable influence on T1 and T2 relaxation rates of the surrounding water protons.
  • U.S. Patent 4,899,755 discloses a method of alternating the proton NMR relaxation times in the liver or bile duct of an animal using Fe +3 -ethylene-bis(2-hydroxyphenylglycine) complexes and its derivatives, and suggests among various other compounds the possible use of a pyridine macrocyclomethylenecarboxylic acid.
  • U.S. Patent 4,880,008 (a CIP of U.S. Patent 4,899,755) discloses additional imaging data for liver tissue of rats, but without any additional complexes being shown.
  • Patent 4,980,148 disclose gadolinium complexes for MRI which are non-cyclic compounds.
  • C. J. Broan et al., J. Chem. Soc, Chem. Commun., 1739-1741 (1990) describe some bifunctional macrocyclic phosphinic acid compounds.
  • C. J. Broan et al., J. Chem. Soc, Chem. Commun., 1738-1739 (1990) describe compounds that are triazabicyclo compounds.
  • I. K. Adzamli et al., J. Med. Chem. 32, 139-144 (1989) describes acyclic phosphonate derivatives of gadolinium complexes for NMR imaging.
  • the only commercial contrast agents available in the U.S.A. are the complex of gadolinium with diethylenetriaminepentaacetic acid (DTPA-Gd + 3 -MAGNEVISTTM by Schering AG) and a D03A derivative [1 ,4,7-tris(carboxymethyl)-10-(2-hydroxypropyl)-1 ,4,7,10-tetraazacyclododecanato]gadolinium (PROHANCETM by Squibb).
  • MAGNEVISTTM and PROHANCETM are each considered as a non-specific/perfusion agent since it freely distributes in extracellular fluid followed by efficient elimination through the renal system.
  • MAGNEVISTTM has proven to be extremely valuable in the diagnosis of brain lesions since the accompanying breakdown of the blood/brain barrier allows perfusion of the contrast agent into the affected regions.
  • Guerbet is commercially marketing a macrocyclic perfusion agent (DOTAREMTM) which presently is only available in Europe.
  • PROHANCETM is shown to have fewer side effects than MagnevistTM.
  • a number of other potential contrast agents are in various stages of development.
  • bicyclopolyazamacrocyclophosphonic acid ligands can be contrast agents.
  • these ligands may have their charge modified, i.e. bythe structure of the ligand and metal selected, which can effect their ability to be more site specific.
  • the present invention is directed to novel ligands that are bicyciopolyazamacrocyclophosphonic acid compounds of the formula
  • X and Y are independently H, OH, C 1 -C 3 alkyl or COOH; n is an integer of 1 , 2 or 3; with the proviso that: when n is 2, then the sum of X and Y must equal two or more H; and when n is 3, then the sum of X and Y must equal three or more H;
  • T is H, C 1 -C 18 alkyl, COOH, OH, SO 3 H,
  • R 1 is OH, C 1 -C 5 alkyl or -O-(C 1 -C 5 alkyl);
  • R 4 is H, NO 2 , NH 2 , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl ;
  • R 2 is H or OH; with the proviso that when R 2 is OH, then the Rterm containing the R 2 must have all X and Y equal to H; with the proviso that at least one T must be P(O)R 1 OH, and with the proviso that when one T is
  • one X or Y of that R term may be COOH and all other X and Y terms of that R term must be
  • A is CH, N, C-Br, C-CI, C-OR 3 , C-OR 8 , N + -R 5 X-,
  • R 3 is H, C 1 -C 5 alkyl, benzyl, or benzyl substituted with at least one R 4 ;
  • R 4 is def i ned as above
  • R 5 is C 1 -C 16 alkyl, benzyl, or benzyl substituted with at least one R 4 ;
  • R 8 is C 1 -C 16 alkylamino
  • X is CI-, Br-, I-or H 3 CCO 2 -;
  • Q and Z independently are CH, N, N + -R 5 X-, C-CH 2 -OR 3 or C-C(O)-R 5 ;
  • R 5 is defined as above;
  • R 6 is -O-(C 1 -C 3 alkyl), OH or NHR 7 ;
  • R 7 is C 1 -C 5 alkyl or a biologically active material
  • X- is defined as above; or pharmaceutically-acceptable salts thereof; with the proviso that: a) when Q, A or Z is N or N + -R 5 X-, then the other two groups must be CH; b) when A is C-Br, C-CI, C-OR 3 or C-OR 8 , then both Q and Z must be CH; c) the sum of the R 4 , R 7 and R 8 terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R 6 and when one of Q or Z is C-C(O)-R 6 , then A must be CH.
  • the above ligands of Formula (I) have at least two of the R terms T equal to PO 3 H 2 [P(O)R 1 OH where R 1 is OH] and the third T equal H, COOH or C 1 -C 18 alkyl; A, Q and Z are CH; n is 1 ; and X and Y independently are H or C 1 -C 3 alkyl; then the ligands are useful for oral care. Particularly preferred are those ligands where in the three R terms T is P(O)R 1 OH, where R 1 is OH; n is 1 ; and X and Y are H. The use of these ligands is discussed and claimed in other copending applications.
  • the above ligands of Formula (I) have: in the Rterm at least two T equal P(O)R 1 OH, where R 1 is OH, and in the other R term, T is COOH or P(O)R 1 OH, and n, R 1 , X, Y, A, Q and Z are defined as above; in at least one R term T is P(O)R 1 OH, where R 1 is OH, and in the other two R terms, T is COOH or P(O)R 1 OH, and n, R 1 , X, Y, A, Q and Z are defined as above; or in the R term three T equal P(O)R 1 OH, where R 1 is C 1 -C 5 alkyl or -O-(C 1 -C 5 alkyl), and n, R 1 , X, Y, A, Q and Z are defined as above; then the ligands are useful as contrast agents. Particularly preferred are those ligands of Formula (I) where: X and
  • the ligands and complexes of Formula (I) do not have all three T equal to PO 3 H 2 [P(O)R 1 OH where R 1 is OH] when A, Q and Z are CH; although such complexes are useful as contrast agents or oral care agents.
  • the ligands and complexes of Formula (I) may have a proviso that not all T may be equal to PO 3 H 2 [P(O)R 1 OH where R 1 is OH] when A, Q and Z are CH, unless used as a contrast agent or oral care agent.
  • Bifunctional ligands of Formula (I) are desirable to prepare the conjugates of this invention. Such ligands must have: one R term where the T moiety is
  • both T terms are P(O)R 1 OH, where R 1 is defined as above or where in the two R terms not containing an R 4 term, one T term is a COOH and the other T term is P(O)R 1 OH, where R 1 is defined as above; preferrably that moiety of the above T term where one of X or Y of that term is COOH; and also preferred are those ligands where n is 1 and/or the remaining X and Y terms are H; or
  • A is C-OR 3 or C-OR 8 , where R 3 and R 8 are defined as above or
  • R 4 is defined as above;
  • A is CH, and one of Q or Z is CH and the other is C-C(O)-R 6 , where R 6 is defined as above; especially those ligands where R 6 is NHR 7 , where R 7 is a biologically active material
  • the ligands of Formula (I) may be complexed with various metal ions, such as gadolinium (Gd + 3 ), iron (Fe + 3 ), and manganese (Mn +2 ), with Gd +3 being preferred.
  • the complexes so formed can be used by themselves or can be attached, by being covalently bonded to a larger molecule such as a dextran, a polypeptide or a biologically active molecule, including an antibody or fragment thereof, and used for diagnostic purposes.
  • Such conjugates and complexes are useful as contrast agents.
  • the complexes and conjugates of Formula (I) can be designed to provide a specific overall charge which advantageously influences the in vivo biolocalization and image contrast. For example, when the metal ion is + 3 the following can be obtained:
  • Both the complexes and conjugates may be formulated to be in a pharmaceutically acceptable form for administration to an animal.
  • One aspect of the present invention concerns development of contrast agents having synthetic modifications to the paramagnetic chelate enabling site specific delivery of the contrast agent to a desired tissue.
  • the advantage being increased contrast in the areas of interest based upon tissue affinity as opposed to contrast arising from non-specific perfusion which may or may not be apparent with an extracellular agent.
  • the specificity of the ligand of Formula (I) may be controlled by adjusting the total charge and lipophilic character of the
  • the overall range of the charge of the complex is from -3 to + 1.
  • the overall charge is highly negative and bone uptake is expected; whereas when the overall charge of the complex is 0 (thus neutral), the complex 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 occurring or synthetic molecule having specificity for a desired target tissue.
  • a paramagnetic chelate to a macromolecule can further increase the contrast agent efficiency resulting in improved contrast relative to the unbound chelate.
  • Lauffer U.S. Patents 4,880,008 and 4,899,755 has demonstrated that variations in lipophilicity can result in tissue-specific agents and that increased iipophilic character favors non-covalent interactions with blood proteins resulting in enhancement of relaxivity.
  • the present contrast agents of Formula (I) which are neutral in charge are particularly preferred for forming the conjugates of this invention since undesirable ionic interactions between the chelate and protein are minimized which preserves the antibody immunoreactivity. Also the present neutral complexes reduce the osmolarity relative to DTPAGd +3 , which may alleviate the discomfort of injection.
  • a charged complex of the invention e.g. possibly -2 or -3 for bone, -1 for liver, or + 1 for heart
  • the variations in that chelate ionic charge can influence biolocalization.
  • 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.
  • C 1 -C 3 alkyl include both straight and branched chain alkyl groups.
  • An “animal” includes a warmblooded mammal, preferably a human being.
  • Biologically active material refers to 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 or epitopes.
  • antibody fragment includes Fab fragments and F(ab') 2 fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes.
  • Possible antibodies are 1 1 16-NS-19-9 (anti-col orectal carcinoma), 1 116-NS-3d (anti-CEA), 703D4 (anti-human lung cancer), 704A1 (anti-human lung cancer), CC49 (anti-TAG-72), CC83 (antiTAG-72) and B72.3.
  • hybridoma cell lines 1 1 16-NS-19-9, 1 1 16-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.
  • complex refers to a complex of the compound of Formula (I) complexed with a metal ion, where at least one metal atom is chelated or sequestered;
  • conjuggate refers to a metal ion chelate that is covalently attached to an antibody or antibody fragment.
  • bifunctional coordinator refers to compounds 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 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 of the presence of the functionalizing moiety (represented by R 4 or R 8 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.
  • biologically active materials such as dextran, molecules that have specific affinity for a receptor, or preferably covalently attached to antibodies or antibody fragments.
  • conjugates are referred to herein as "conjugates”.
  • salts means any salt or mixtures of salts of a compound of Formula (I) which is sufficiently non-toxic to be useful in therapy or diagnosis of animals, preferably mammals. Thus, the salts are useful in accordance with this invention.
  • 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, palmoic, mucic, glutamic, gluconic acid, d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic acids and other suitable acids.
  • salts formed by standard reactions from both organic and inorganic sources such as ammonium or 1-deoxy-1-(methylamino)-D-glucitol, 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. Also included are mixtures of the above salts.
  • T PO 3 H 2
  • Q, A and Z CH.
  • R 1 C 1 -C 5 alkyl
  • Q, A and Z CH.
  • R 1 -O-(C 1 -C 5 alkyl) or C 1 -C 5 alkyl;
  • Q and Z CH.
  • R 1 -OH, -O-(C 1 -C 5 alkyl) or C 1 -C 5 alkyl;
  • R 1 -OH, -O-(C 1 -C 5 alkyl) or C 1 -C 5 alkyl
  • Q and A CH.
  • R 1 -OH, -O-(C 1 -C 5 alkyl) or C 1 -C 5 alkyl
  • A N or N-R 5
  • R 5 C 1 -C 16 alkyl halide
  • Q and Z CH.
  • R 1 -OH, -O-(C 1 -C 3 alkyl) or C 1 -C 5 alkyl
  • Q N-R 5
  • R 5 C 1 -C 16 alkyl halide
  • a and Z CH.
  • R 1 -OH, -O-(C 1 -C 5 alkyl) or C 1 -C 5 alkyl;
  • Q N or N-R 5 ,
  • R 5 C 1 -C 16 alkyl halide; and
  • AandZ CH.
  • the synthetic Scheme 1 begins with a halogenation 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 HCl, 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 macrocycles using milder conditions [A. D. Sherry et al., J. Org. Chem.
  • phosphonate esters [e.g. of formula (6)] can also be prepared under organic conditions in alcohols or aprotic solvents (e.g. acetonitrile, benzene, toluene, tetrahydrofuran) and using the desired dialkyl phosphite as the nucleophilic species (see Scheme 2). Depending upon the reactivity of the ami ne, these reactions may be conducted at a temperature between about -10 to about 100°C.
  • alcohols or aprotic solvents e.g. acetonitrile, benzene, toluene, tetrahydrofuran
  • trialkylphosphites can be employed under similar Mannich conditions to give the phosphonate ester via oxidation of phosphorous (III) to phosphorous (V) with simultaneous expulsion of one mole of alcohol (Arbuzov reaction). These reactions can be conducted with or without the presence of a solvent.
  • alcohols are employed as the solvent for either dialkyl or trial kyl phosphite reactions, it is beneficial to use the alcohol from which the corresponding phosphonate ester is derived in order to avoid alternative products arising from transesterification.
  • Esters of this type are also prepared via N-alkylation of ⁇ -halodialkylphosphonates in solvents such as acetonitrile, chloroform, dimethylformamide, tetrahydrofuran or 1,4-dioxane with or without the addition of a non-nucleophilic base such as potassium carbonate at room temperature or above.
  • solvents such as acetonitrile, chloroform, dimethylformamide, tetrahydrofuran or 1,4-dioxane
  • a non-nucleophilic base such as potassium carbonate at room temperature or above.
  • macrocyclic methylphosphinic acids (10 and 1 1) are prepared under conditions similar to those described in Scheme 2.
  • condensation can be conducted in solvents such as tetrahydrofuran, dimethylformamide, dioxane, acetonitrile or alcholic media.
  • the resulting phosphi ⁇ ate ester is then hydrolyzed under acid (6N HCl, 80-100°C) or basic (stoichiometric quantities of base, 40-100°C) conditions to give the corresponding methyl phosphonic acid.
  • the method devised by A. D. Sherry et al. ⁇ Inorg. Chem., submitted 1991) using ethylphosphonic acid generated in situ can be used to obtain phosphinate derivatives having increased lipophilic character.
  • Scheme 4 illustrates an approach to incorporate additional functionality into the pyridine unit of the 12-membered tertaazamacrocycle.
  • chelidamic acid Sigma Chemical Company; 12
  • the bis-halomethyl derivative (13) having appropriate substitution at the pyridyl 4-position. Transformations leading to this intermediate are general in nature and its preparation is described by Takalo et al. [Ada Chemica Scandinavica B 42, 373-377(1988)].
  • Subsequent macrocyclization using this intermediate (15) can be accomplished by the standard DMF reaction at 100°C with the sodiotritosylated triamine, or at room temperature with the tritosylated free base and potassium carbonate, sodium carbonate, or cesium carbonate as base to give products similar to those previously described. Subsequent reactions leading to phosphonate half-acids and phosphinate functionality are identical to those transformations and conditions described in the preceeding Schemes.
  • organometallic Pd(ll) complexes can be employed to facilitate the coupling reaction between phenylacetylene and phenylacetylene derivatives and the pyridyl macrocycle.
  • Typical reaction conditions for this transformation utilize anhydrous conditions with triethylamine as solvent and at reaction temperature between about 10 to about 30°Cfor optimum yields.
  • the identical product can also be obtained using Cu(l) phenylacetyiide in anhydrous pyridine at a temperature between about 80 to about 1 10°C.
  • the nicotinic acid macrocycle can then be substituted into the general scheme for secondary amine functionalization to yield the various types of phosphonate chelants of Formula (I) (49, 50, 51 , 52, 53).
  • the 3-hydroxymethyl analog is advantageously protected prior to functionalization of the macrocyclic amines.
  • the benzyl (Bz) protecting group is shown in Scheme 8 since it must be resistant to the severe acid conditions encountered in the detosylation step. After appropriate functionalization of the secondary amines has been accomplished as described in previous Schemes, the benzyl group is removed under mild catalytic hydrogenation conditions (58).
  • Macrocyclic derivatives can also be prepared as in Schemes 12-14 where both carboxylate and phosphonate chelating fuctionalities are present in the same molecule.
  • carboxylate fuctionality can be introduced under typical aqueous alkylation procedures using bromoaceticacid.
  • the remaining amines can be phosphonomethylated by procedures discussed in previous Schemes using formaldehyde and phosphorous acid, dialkyl phosphonates or trialkyl phosphites.
  • Schemes 15 and 16 delineate a synthetic approach which introduces an aromatic nitrobenzyl substitutent at one of the macrocyclic nitrogen positions.
  • the macrocyclic amine is mono-N-functionalized in an organic solvent such as acetonitrile or DMF at room temperature using a non-nucleophilic base such as potassium carbonate. Additional functionalization of the remaining nitrogen positions is then performed by methodsand conditions described in previous Schemes.
  • the nitro group is reduced using platinum oxide and hydrogen in water.
  • the chelating agent is compatible with conjugation techniques which will enable attachment to larger synthetic or natural molecules.
  • Scheme 17 illustrates the synthesis of the macrocyclic compounds (4) where the amines at positions 3 and 9 are reacted with at least two moles of the sodium salt of hydroxymethanesulfonic acid in water at a pH of about 9 to provide the corresponding macrocyclic compound where positions 3 and 9 are the sodium salt of methanesulfonic acid (119). The sulfonic acid group is then displaced using sodium cyanide to form the corresponding cyanomethane derivative (120).
  • the cyano group is hydrolyzed to the carboxylic acid either: simultaneously with the addition of phosphorous acid and formaldehyde; or by sequential reaction with a derivative of phosphorous acid and formaldehyde to form the phosphonic acid at the 6 position (121), followed by acid hydrolysis, at an elevated temperature, of the cyanato groups and any derivative moiety of the phosphorous acid present.
  • the resulting compound is a macrocycle with two carboxylic acid groups at positions 3 and 9 and a phosphonic acid group at position 6.
  • the phosphonomethylation can also be preformed by the methods discussed above.
  • the metal ions used to form the complexes of this invention are Gd +3 , M n +2 , Fe + 3 and available commercially, e.g. from Aldrich Chemical Company.
  • the anion present is halide, preferrably chloride, or salt free (metal oxide).
  • a "paramagnetic nuclide” of this invention means a metal ion which displays spin angular momentum and/or orbital angular momentum.
  • the two types of momentum combine to give the observed paramagnetic moment in a manner that depends largely on the atoms bearing the unpaired electron and, to a lesser extent, upon the environment of such atoms.
  • the paramagnetic nuclides found to be useful in the practice of the invention are gadolinium (Gd + 3 ), iron (Fe + 3 ) and manganese (Mn +2 ), with Gd +3 being preferred.
  • 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.
  • the complexes of the 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, Kohlerand Milstein [Nature. 256, 495-497 (1975); and Eur. 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 1 1 16-NS-19-9, 1 1 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 therefore.
  • 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" of the formulation is used for diagnosis.
  • the dose will vary depending on the disease and physical parameters of the animal, such as weight.
  • In vivo diagnostics are also contemplated using formulations of this invention.
  • chelants of the present invention may include the removal of undesirable metals (i.e. iron) from the body, attachment to polymeric supports for various purposes, e.g. as diagnostic agents, and removal of metal ions by selective extraction.
  • the ligands of Formula (I) having in at least two R terms T equal to P(O)R 1 OH may be used for metal ion control as scale inhibitors. Some of these ligands can be used in less than stoichiometric amounts. Similar uses are known for compounds described in U.S. Patents 2,609,390; 3,331 ,773; 3,336,221 ; and 3,434,969.
  • ICP inductively coupled plasma.
  • mL milliliter(s).
  • ⁇ L microliter(s).
  • a stock 159 GdCI 3 (or 153 SmCI 3 ) solution was prepared by adding 2 ⁇ L of 3 ⁇ 10 -4 M 159 GdCI 3 in 0.1 N HCl to 2 mL of a 3 ⁇ 10 -4 M GdCI 3 carrier solution.
  • Appropriate ligand solutions were then prepared in deionized water.
  • the percent metal as a complex was then determined by passing a sample of the complex solution through a SephadexTM G-50 column, eluting with 4: 1 saline (85% NaCI/NH 4 OH) and collecting 2 ⁇ 3 mLfractions. The amount of radioactivity in the combined elutions was then compared with that left on the resin (non- complexed metal is retained on the resin).
  • the pH stability profile was generated by adjusting the pH of an aliquot of the complex solution using 1 M NaOH or 1M HCl and determining the percent of the metal existing as a complex using the ion exchange method described above. The Sm results are known by expermintal comparison to be identical for complexation and biodistribution of the ligands of this invention. STARTING MATERIALS Example A Preparation of 2,6-bis(chloromethyl)pyridine.
  • a DM F solution (92 mL) of 6.9 g (1 1.4 mmol) of 1 ,4,7-tris(p-tolylsulfonyl)diethylenetriamine disodium salt was stirred and heated to 100°C under nitrogen.
  • To the solution was added dropwise over 45 min 2 g (1 1.4 mmol) of 2,6-bis(chloromethyl)pyridine (prepared by the procedure of Example A) in 37 mL of DMF.
  • the reaction mixture was stirred at40°C for 12 hrs.
  • Asolution of HBr and AcOH was prepared by mixing 48% HBr and glacial AcOH in a 64:35 ratio.
  • To 1 12 mL of the HBr/AcOH mixture was added 5.5 g (8.2 mmol) of 3,6,9-tris(p-tolyls ⁇ lfonyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),1 1 ,13-triene (prepared by the procedure of Example B) and the reaction mixture was heated at mild reflux with constant stirring for 72 hrs. The reaction mixture was then cooled to room temperature and concentrated to approximately 1/10 of the original volume. The remaining solution was stirred vigorously and 15-20 mL of diethyl ether was added.
  • Example C 318 mg (1.53 mmol) of tripropyl phosphite and 46 mg (1.53 mmol) of paraformaldehyde.
  • the reaction mixture was heated at 90°C with stirring for 1 hr.
  • the resulting homogenous solution was concentrated in vacuo to give a viscous oil which was chromatographed on a neutral alumina column, eluting with chloroform.
  • a 1 : 1 ligand/metal complex was then prepared by combining 40 ⁇ l of the ligand solution with 2 mL of aqueous SmCI 3 ⁇ H 2 O (3 ⁇ 10 -4 M in 0.01 N HCl) contai ning tracer 153 SmCI 3 .
  • the percent metal as a complex was determined by passing a sample ofthe complex solution through a SephadexTM column, eluting with 4: 1 saline (0.85% NaCI/NH 4 OH), and collecting 2 ⁇ 3 mL fractions.
  • Sprague Dawley rats were allowed to acclimate for five days then injected with 100 ⁇ L of the complex solution via a tail vein.
  • the rats weighed between 150 and 200 g at the time of injection. After 30 min. the rats were killed by cervical dislocation and dissected.
  • the amount of radioactivity in each tissue was determined by counting in a Nal scintillation counter coupled to a multichannel analyzer. The counts were compared to the counts in 100 ⁇ L standards in order to determine the percentage of the dose in each tissue or organ.
  • the percent dose in blood was estimated assuming blood to be 7% ofthe body weight.
  • the percent dose in bone was estimated by multiplying the percent dose in the femur by 25.
  • the percent dose in muscle was estimated assuming muscle to be 43% of the body weight.
  • chelates of the compounds of Formula (I) were evaluated for efficiency of bone localization since phosphonates are known fortheir ability to bind to hydroxyapatite.
  • the percent of the injected dose of complex of of of Example 2 ( 153 Sm-PCTMP) in several tissues are given in Table I.
  • the numbers represent the average of a minimum of 3 rats per data point at 2 hours ost in ection.
  • the percent of the injected dose of complex of of of Example 5 ( 153 Sm-PMPHE) in several tissues are given in Table II.
  • the numbers represent the average of a minimum of 3 rats per data point at 2 hours post injection.
  • EXAMPLE III The percent of the injected dose of complex of of Example 6 ( 153 Sm-PMBHE) in several tissues are given in Table III. The numbers representthe average of a minimum of 3 rats per data point at 2 hours post injection.
  • the percent ofthe injected dose of complex of of of Example 3 ( 153 Sm-PC2A1) in several tissues are given in Table IV.
  • the numbers representthe average of a minimum of 3 rats per data point at 2 hours post injection.
  • Injectable solutions were first prepared (0.5M) by dissolving the appropriate amount of each complex in 2 mL of deionized water. The pH of the solutions were then adjusted to 7.4 using 1M HCl or NaOH as needed. The total Gd content of each solution was then determined by ICP analysis.
  • the Gd-PCTMP complex (prepared in Example 2) showed kidney enhancement and bone localization in the shoulder, spine and sternum.

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Abstract

Composés d'acides bicyclopolyazamacrocyclophosphoniques pouvant former des complexes inertes avec des ions Gd, Mn ou Fe. La charge globale du complexe peut être modifiée pour changer la biolocalisation in vivo. Ces complexes peuvent être liés par covalence à un anticorps, un fragment d'anticorps ou une autre molécule biologiquement active pour former des conjugués. Les complexes et les conjugués sont utiles comme agents de contraste dans des applications de diagnostic. Des procédés de préparation du ligand, du complexe et du conjugué sont également décrits.
EP93911136A 1993-05-06 1993-05-06 Acides bicyclopolyazamacrocyclophosphoniques, leurs complexes et conjugues, destines a etre utilises en tant qu'agents de contraste, et leurs procedes de preparation Withdrawn EP0696290A1 (fr)

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US6207826B1 (en) 1995-03-27 2001-03-27 Isis Pharmaceuticals, Inc. Macrocyclic compounds having nitrogen-containing linkages
WO1996030377A1 (fr) * 1995-03-27 1996-10-03 Isis Pharmaceuticals, Inc. Composes macrocycliques azotes
CA2251924C (fr) * 1996-04-19 2006-05-30 The Dow Chemical Company Chelates fluorescents utilises en tant qu'agents visuels destines a l'imagerie et specifiques de certains tissus
US6005083A (en) * 1997-03-28 1999-12-21 Neorx Corporation Bridged aromatic substituted amine ligands with donor atoms
US6776977B2 (en) 2001-01-09 2004-08-17 Bristol-Myers Squibb Pharma Company Polypodal chelants for metallopharmaceuticals
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FR2644785B1 (fr) * 1989-03-24 1991-07-05 Guerbet Sa Nouveaux ligands macrocycliques azotes, procede de preparation, complexes metalliques formes par ces ligands et composition de diagnostic les contenant
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NO954442D0 (no) 1995-11-06
AU4238293A (en) 1994-12-12
NO932823D0 (no) 1993-08-09
JPH08509976A (ja) 1996-10-22
NZ252554A (en) 1998-01-26
NO304985B1 (no) 1999-03-15
NO954442L (no) 1996-01-05
KR960702470A (ko) 1996-04-27
FI933507A (fi) 1994-11-07
FI955335A0 (fi) 1995-11-06
AU665689B2 (en) 1996-01-11

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