BICYCLE-POLYAZAMACROCYCLOCARBOXYLIC ACID COMPLEXES, CONJUGATES, PREPARATION AND USE AS CONTRAST AGENTS

Abstract: Complexes of bicyclopolyazamacrocyclocarboxylic acid with Gd, Mn or Fe ions 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 contrast agents for diagnostic purposes. Processes for preparing both the complex and conjugate are disclosed.

Description: 'Bicycle-Polyazamacrocyclocarboxlic Acid Complexes , Conjugates , Preparation and Use as Contrast Agents" This invention concerns complexes that contain as the ligand bicyclopolyaza- macrocyclocarboxylic acids, and conjugates thereof, for use as contrast agents in magnetic resonance imaging ( RI). Processes for preparing these complexes and conjugates are also provided. To better understand this invention, a brief background on MRI is provided in the following section. Background MRI is a non-invasive diagnostic technique which produces well resolved cross- sectional images of softtissuewithin 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. Barrow, Physical Chemistry, 3rd Ed., McGraw-Hill, NY (1973)] to align in an applied magne* c field. Once aligned, this equilibrium state can be perturbed by applying an external radio frequency (RF) pulse which causes the protons to be tilted out of alignment with the magnetic field. When the RF pulse is terminated, the nuclei return to their equilibrium state and the time required for this to occur is known as the relaxation time. 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. Since water content of living tissue is substantial and variations in content and environment exist among tissue types, diagnostic images of biological organisms are obtained which reflect proton density and relaxation times. The greater the differences in relaxation times (T1 and T2) of protons present in tissue being examined, the greater will be the contrast in the obtained image [J. Magnetic Resonance 33, 83-106 (1979)]. It is known that paramagnetic chelates possessing a symmetric electronic ground state can dramatically affect the T1 and T2 relaxation rates of juxtaposed water protons ar> thatthe effectiveness of the chelate in this regard is related in part, to the number of unp _ red electrons producing the magnetic moment [Magnetic Resonance Annual 231 -266 (Raver Pres. 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 inthe magnetic resonance (MR) images with increased contrast being observed in the areas of chelate localization. It has therefore been proposed that stable, non-toxic paramagnetic chelates be administered to animals in order to increase the diagnostic information obtained by MRI [Frontiers of Biof. Energetics 1752-759 (1978); J. Nucl. Med. 25, 506-513 (1984); Proc. of NMR Imaging Svmp. (Oct.26-27, 1980); F. A. Cotton eta , Adv. Inorg. Chem.634-639 (1966)]. Paramagnetic metal chelates used in this manner are referred to as contrast enhancement agents or contrast agents. There are a number of paramagnetic metal ions which can be considered when undertaking the design of an MRI contrast agent. In practice, however, the most useful paramagnetic metal ions are gadolinium (Gd * 3 ), iron (Fe +3 ) r 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. In a non-complexed form (e.g. GdCI 3 ), 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. Art in the MRI field is quite extensive, such that the following summary, not ~~ intended to be exhaustive, is provided only as a review of this area and other compounds that are possibly similar in structure. 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 acϊd. 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. U.S. Patent 4,980,148 disclose gadolinium complexes for MRI which are non-cyclic compounds. C J. Broan etal., J. Chem. Soc. r Chem. Commun., 1739-1741 (1990) describe some bifunctional macrocyclic phosphinic acid compounds- C. J. Broan etal., J. Chem. Soc. r Chem. Commun., 1738-1739 (1990) describe compounds that are triazabϊcyclo compounds- I- K. Adzamli etal.,i. Med. Chem. 32, 139-144 (1989) describes acyclic phosphonate derivatives of gadolinium complexes for N R imaging. At the present time, the only commercial contrast agent available in the U.S. is the complex of gadolinium with diethylenetriarninepentaacetic acid (DTP A-Gd *3 - MAGNEVIST™ * byShering). MAGNEVIST™ is considered as a non-specific/perfusion agent since it freely distributes in extracellular fluid followed by efficient elimination through the renal system MAGNEVIST™ 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, in addition to MAGNEVIST'", Guerbet is commercially marketing a macrocyclic perfusion agent (DOTAREM™) which presently is only available in Europe. A number of other potential contrast agents are in various stages of development. It would be advantageous if contrast agents were developed that could have site specificity forthe tissue desired tt. be imaged, rather than non-specific/perfusion agents. The present invention is directed to just such novel complexes comprising a ligand that is a bicyclo- polyazamacrocyclocarboxylic acid of the formula wherein: R is X X where: X and Y 1 are independently H, OH or C t -C- alkyl; Y 2 is H or COOH; R 7 is H, OH or OCH 3 ; and R 4 is H, N0 2 , NH 2 , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl; with the proviso that at least two R terms must be -C-C0 2 H A = CH, N, C-Br, C-CI, C-OR 1 , C-OR 2 , N + -R 3 X " , or R 1 = H, C.-C 3 alkyl, benzyl, or benzyl substituted with at least one R 4 ; R 2 is C -C 16 alkylamino; R 3 is C -C alkyl, benzyl, or benzyl substituted with at least one R 4 ; R d is defined as before; X- isC! ' , Br-, !-orH.CCO--; Q and Z independently are CH, N, N + -R 3 X * , C-CH^OR 1 or C-C(0)-R 5 ; R 1 and R 3 are defined as above; R s is-0-(C,-C 3 alkyl), OH or NHR 6 ; R 6 is C -C alkyl or a biologically active material; X ' is defined as above; and with the provisos that: a) when Q, A or Z is N or N + -R 3 X " , then the other two groups must be CH; b) when A is C-Br, C-CI, C-OR 1 or C-OR 2 , then both Q and Z must be CH; c) the sum of the R 2 , R 4 and R ε terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(0)-R s and when one of Q or Z is C-C(0)-R 5 , then A mustbe CH; and complexed with a metal ion selected from Gd +3 , Mn *2 or Fe +3 ; or pharmaceutically-acceptable salts thereof. Bifunctional complexes of Formula (I) are desirable to prepare the conjugates of this invention. Such ligands must have at least one of R 2 , R 4 or R s present: one R term is -A- where X, Y 2 , R 4 and R 7 are defined as above; or A is C-OR 1 , C-OR 2 , where R 1 and R 2 are defined as above or where R 4 is defined as above; or A is CH, and one of Q or Z is CH and the other is C-C(0)-R 5 or C-CH 2 -OR\ where R 1 and R 5 are defined as above; especially perferred are those ligands where R 5 is NHR 6 , where R 6 is a biologically active material. " ~ The ligands of Formula (I) are complexed with various metal ions, such as gadolinium (Gd +3 ), iron (Fe +3 ), and manganese (Mn +2 ), and 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 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 R is X I -C-C0 2 H I γ ι and X and Y 1 are all equal to H; (B) an overall + 1 charge - when one of A, Q or Z is N + -R 3 X ~ , where R 3 and X ' are defined as above; and the three R terms are X I 5 -C-C0 2 H ; i Y 1 and X and Y 1 are all equal to H. 10 Both the complexes and conjugates may be formulated to be in a pharmaceutically acceptable form for ad ministration to an animal. Use of the 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 copendϊng application. T5 The complex has the ligand of Formula (I) numbered for nomenclature purposes as follows: 13 5 R The present invention concerns development of contrast agents having a neutral or + 1 charge which enablessitespecifϊc deliveryof the contrastagentto 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 0 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 complex. The overall range of the charge of the complex is from + 1 to 0. For example, for a complex having a + 1 overall charge has heart uptake 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 5 be possible. Tissue specifi ity may also be realized by ionic or covalent attachment of the chelate to a naturally occurring or synthetic macromoiecule having specificity for a desired target tissue. One possible application of this approach isthrough the use of chelate conjugated monoclonal antibodies which would transport the paramagnetic chelate to diseased tissue enabling visualization by MRI. In addition, attachment of a paramagnetic chelate to a macromolecule can further increase contrast agent efficiency resulting in improved contrast relative to the unbound che Recent work by Lauffer (U.S. Patents 4,880,008 and 4,899,755) has demonstrated tha 1 iations in lipophilicity can result in tissue- specific agents and that increased lipophilic cha J ..er favors non-covalent interactions with blood proteins resulting in enhancement of relaxivity. Additionally, 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 DTPA- Gd +3 , which may alleviate the discomfort of injection. While not wishing to be bound by theory, it is believed that when 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 for this invention are further defined as follows. "C C 3 alkyl", "C C 5 alkyl", "C,-^, alkyl", inclu 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 or epitopes. 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 1 116-NS-19-9 (anti-colorectal carcinoma), 1 1 16-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 hybridoma cell lines 1116-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. As used herein, "complex" refers to a complex of the compound of the 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 antibody or antibody fragment. The terms "bifunctional coordinator", "bifunctional chelating agent" and "functionalized chelant" are used interchangeably and referto compounds that have a chelant moiety capable of chelating a metal ion and a moiety covalently bonded to the chelant moiety that ϊscapable of serving as a means to covalently attach to an antibody or anti body frag ment. 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 of the presence of the functionalizing moiety (represented by R2, *or R6jn Formula I), can be covalently attached to biologically active materials, such as dextran, molecules that have specific affinity for a receptor, or preferably covalentlyattached 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 therapyordiagnosis of animals, preferably mammals. Thus, the salts are useful in accordance with this invention. Representative of those salts formed by standard reactions from both organicand inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, muck, glutamic, gluconic, d- camphorϊc, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic? " benzenesuifonic, sorbic, picric, benzoic, cinnamic acids and other suitable acids. Also included are salts formed by standard reactions from both organic and inorganic sources such as ammonium or l-deoxy-1-(methylamino)-D-glucitol, 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 (1). 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 I -C-C0 2 H • Y 1 Scheme 1 Scheme 2 preparesthe compounds of Formula (I) wherein A = C-Br, and Q and Z = CH- Scheme 2 (9) (10) (11) Scheme 2 Cont'd Br I H t a coumpound of (11) I Formula (I) Scheme 3 preparesthe compounds of Formula (I) wherein A = R 4 = H, N0 2 , NH 2 or SCN; and Q and Z = CH. Scheme 3 (13) (14) Scheme 3 Cont'd (15) a compound of Formula (I) Scheme 4 preparesthe compounds of Formula (I) wherein A = C-OR 2 , where R z - C -C,. alkylamino; and Q and Z = CH. Scheme 4 Cont'd Scheme 4 Cont'd (26) a compound of Formula (I) Scheme 5 preparesthe compounds of Formula (I) wherein A = CH; and one of Q the other Q or Z = C-C(0)-R 6 or C-CH 2 -R 6 , where R 6 is defined as before. Scheme 5 Scheme 5 Cont'd (32) a compound of Formula (I) Scheme 6 preparesthe compounds of Formula (I) wherein Z = C-CH 2 -OBz or C- C(0)-R s where R 5 = -O-tC,-^ alkyl), OH or NHR 6 , where is defined as before; and Q and A = CH. Scheme 6 a compound of Formula (I) Scheme 6 Cont'd I rO LΠ I a compound of Formula (I) Scheme 7 preparesthe compounds of Formula (I) wherein A = Nor * -R 5 X ' ; R 5 = C-C alkyl and isX ' halide; and QandZ = CH. H 3 C CH 3 (38) Scheme 7 Cont'd Scheme 8 prepares the compounds of Formula (I) wherein Q = N + -R 5 X-, where R 5 = C C 16 alkyl and X " = halide; and AandZ = CH. Scheme 8 Scheme 8 Cont'd 1 10 a compound of Formula (I) I Scheme 9 prepares the compounds of Formula (I) wherein Q = N or N + -R s X " , where R 5 = C 1 -C lo alkyl and .. X " = halide; and Aand Z = CH. Scheme 9 a c a compound of Formula (I) Scheme 10 preparesthe compounds of Formula (I) wherein Rterm atthe 6 positron is where R 4 4 _ = NOpr NH 2 ; Y 2 = CO H (orwith a change of reagent Y 2 = H); and A r Qand Z = CH. Scheme 10 Scheme 10 Cont'd (56) (55) compound of Formula (I) Scheme 11 prepares the compounds of Formula (I) wherein the R term at the 9 position is where R 4 = NO.orNH • Y 2 = CO.H (orwith a change of reagent Y 2 = H); and A,QandZ = CH Scheme 11 (57) (58) compound of Formula (I) a compound of Formula (I) Scheme 12 prepares the compounds of Formula (I) wherein n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), the R term at the 6 position hasT = - °P-OH ; where R 1 = -OH; and X and Y = H; the R term at the 3 and 9 positions have T = COOH; R 7 = OH or OCH 3 ; and A, Q and Z = CH. Scheme 12 Cont'd In the above Schemes, the general process description 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 halogenation of commercially available bis- pyridyl alcohol (1) using thϊonyl 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 similarly reactive product which would work well in subsequent ring closure reactions. Macrocyclization procedures are numerous in the literature and the desired 1f . 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 etal., J. Org. Chem. 54, 2990-2992 (1989)]- Detosylation of the 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, 11 and 12delineate a synthetic approach which introducesan aromatic nitrobenzyl substituent at one of the macrocyclic nitrogen positions. Typically, the macrocyclic amine is mono-N-functionalϊzed in an organic solvent such as acetonitrile or DMF at room temperature using a non-nudeophilic base such as potassium carbonate. Additional -*- _- functionalization of the remaining nitrogen positions is then preformed by methods and conditions described in previous Schemes- After the introduction of the desired chelating moieties, the nitro group is reduced 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 Gd *3 , Mn *2 , Fe +3 and available commercially, e.g. from Aldrich Chemical Company. The anion present is halide, preferably 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 _ n 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. See also methods for making amino acids in Synthetic Production and Utilization of Amino Acids, (edited by Kameko, et al.) John Wiley & Sons (1974). An example of the preparation of a complex involves reacting a bicyclopolyazamacrocydophosphonϊc acid 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 paramagnetic nuclide composition, e.g. stable to the disassociation of the paramagnetic nuclide from the ligand. 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, 95-497 (1975); and Bur. 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 specif icity 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 fragments are 1116-NS-19-9, 1 1 16-NS-3d, 703D4, 704A1, CC49, CC83 " and B72.3. Al I of these anti bod i es have been deposited i n ATCC. A more com pi ete I ist 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" 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. Other uses of some of the chelants of the present i nvention may i ncl ude 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 ion by selective extraction. The ligands of Formula (I) having in at ieast two R terms T equal to P^R'OH may be used for metal ion control as scale inhibitors. It is likely that these ligands could 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. The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the present invention.

Claims: WHAT IS CLAIMED IS: 1. A complex which comprises a bicyclopolyazamacrocyclocarboxylic acid compound of the formula wherein: R is X where: X and Y 1 are independently H, OH or C C_ alkyl; Y 2 is H or COOH; R 7 is H, OH or OCH 3 ; and R 4 is H, N0 2 , NH 2 , isothiocyanato, semicarbazido,thiosemicarbazido, maleimido, bromoacetamido or carboxyl; with the proviso that at least two R terms must be X -C-C0 2 H Y 1 A = CH, N, C-Br, C-CI, C-OR 1 , C-OR 2 , N * -R 3 X ' , or where: R 1 = H, C 1 -C 5 alkyl, benzyl, or benzyl substituted with atfeastone R 4 ; R 2 is C^C^ alkylamino; R 3 is C.,-C alkyl, benzyl, or benzyl substituted with at least one R 4 ; R 4 is defined as before; X is Cr, Br, l-or H 3 CC0 2 -; Q and Z independently are CH, N, N * -R 3 X\ C-CH^OR 1 or C-C(0)-R s ; R 1 and R 3 are defined as above; R s is -0-(C t -C 3 alkyl), OH or NHR 6 ; R 6 is C t -C 3 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 *-R 3 X " , then the other two groups must be CH; b) when A is C-Br, C-CI, C-OR 1 or C-OR 2 , then both Q and Z must be CH; c) the sum of the R 2 , R and R 6 terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(0)-R 5 and when one of Q or Z is C-C(0)-R 5 , then A must be CH; and complexed with a metal ion selected from Gd *3 , Mn *2 or Fe *3 ; or pharmaceutically-acceptable salts thereof. 2- A complex of Claim 1 wherein the metal is Gd *3 . 3. A complex of Claim 1 wherein A, Q and Z are CH; and X and Y are H. 4. A complex of Claim 1 wherein X and Y are H. 5. A complex of Claim 1 wherein A, Q and Z are CH. 6. A complex of Claim 1 wherein Q, A and Z are CH; and one R term is where: X, Y, R 2 and R 4 are defined as in Claim 1. 7. A complex of Claim 1 wherein A is C-OR 1 , C-OR 2 , where R 1 and R 2 are defined as in Claim 1 or where R 4 is defined as in Claim 1. 8. A complex of Claim 1 wherein A is CH, and one of Q or Z is CH and the other is C-C(0)-R 5 or C-CH 2 -OR\ where R 1 and R 5 are defined as in Claim 1. 9. A complex of Claim 8 wherein R 5 is NHR 6 , where R 6 is a biologically active material. 10. A conjugate comprising a bicyclopolyazamacrocyclocarboxylic acid compound of the formula wherein: is X where: and Y 1 are independently H, OH or C t -C 3 alkyl; 15 Y 2 is H or COOH; R 7 is H, OH orOCH 3 ; and R s H, N0 2 , NH 2 , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl; with the proviso that at least two R terms are 20 -C-C0 2 H 25 A = CH, N, C-Br, C-CI, C-OR 1 , C-OR 2 , N*-R 3 X ' , or -JU R 1 = H, C t -C 3 alkyl, benzyl, or benzyl substituted with at least one R 4 ; R 2 is C -C,,. alkylamino; - R 3 is C.-C. B alkyl, benzyl, or benzyl substituted with at least one R 4 ; R 4 is defined as before; X is C|-, Br, l-or H-CCO " ; 5 3 2 Q and Z independently are CH, N, N * -R 3 X " , C-CH^OR 1 or C-C(0)-R s ; R 1 and R 3 are defined as above; R s is -0-(C C 3 alkyl), OH or NHR 6 ; R 6 is C.-C 5 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*-R 3 X " , then the other two groups must be CH; 5 b) when A is C-Br, C-CI, C-OR 1 or C-OR 2 , then both Q and Z must be CH; c) the sum of the R 2 , R 4 and R 6 terms may not exceed one, and one of R 2 , R 4 or R 6 must be present; and d) only one of Q or Z can be C-C(0)-R 5 and when one of Q or Z is C-C(0)-R 5 , then A must be CH; 10 comptexed with a metal ion selected from Gd *3 , Mn *2 or Fe *3 ; and covalently attached to a biologically active material. 11. A conjugate of Claim 10 wherein the biologically active material is a dextran, a peptide, p molecule that has specific affinity for a receptor, or an antibody or ι ς antibody fragment. 12. A conjugate of Claim 1 1 wherein the antibody or antibody fragment is a monoclonal antibody or fragment thereof. 13. A conjugate of Claim 12 wherein the antibody or antibody fragment is B72.3. 14. A conjugate as claimed in any one of Claims 10-13 wherein the metal ion is 0 Gd + 3 15. A conjugate of Claim 10 wherein X and Y are H. 16. A conjugate of Claim 10 wherein A, Q and Z are CH. 17. A conjugate of Claim 10 wherein Q, A and Z are CH; and one Rterm is 5 0 where: X and R 4 are defined as in Claim 10. 18. A conjugate of Claim 10 wherein Q, A and Z are CH; and one Rterm is where: R 4 and R 7 are defined as in Claim 10. 19. A conjugate of Claim 10 wherein A is C-OR 1 , C-OR 2 , where R 1 and 2 are defined as in Claim 10, or where R 4 is defined as in Claim 10. 20. A conjugate of Claim 10 wherein A is CH, and one of Q or Z is CH and the other is C-C(0)-R 6 , where R 6 is defined as in Claim 10- 21. A conjugate of Claim 20 wherein R 6 is NHR 7 , where R 7 is a biologically active material. 22. A pharmaceutical formulation comprising a complex as claimed in any one of Claims 1-9 with a pharmaceutically-acceptable carrier. 23. A pharmaceutical formulation comprising a conjugate as claimed in any one of Claims 10-21 with a pharmaceutically-acceptable carrier. 24. A method for the diagnosis of a disease state in an animal whichTcomprϊses administering to said animal an effective amount of the formulation of Claim 22. 25. A method for the diagnosis of a disease state in an animal which comprises- administering to said animal an effective amount of the formulation of Claim 23. 26- The complex as claimed in an one of Claims 1 -9 for use as a pharmaceutical. 27- The conjugate as claimed in any one of Claims 10-21 for use as a pharmaceutical. 28. A kit for use as a diagnostic agent having as an ingredient a ligand as claimed in any one of Claims 1-9. 29. A process for preparing a complex as claimed in Claim 1 which comprises reacting a bicyclopolyazamacrocyclocarboxyiic acid compound as claimed in Claim 1 with a metal ion selected from Gd *3 , Mn *2 or Fe* 3 underaqueous conditions at a pH from 5 to 7. 30. The process of Claim 29 wherein the bicyclopolyazamacrocyclocarboxylic acid compound is3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-tri- methylenecarboxylic acid.

Inventors: KIEFER GARRY E, SIMON JAIME, KIEFER, GARRY, E., SIMON, JAIME
Applicants: DOW CHEMICAL CO, THE DOW CHEMICAL COMPANY
Application year: 1992
Main classification: C07F15
IPC codes: A61K49, A61K51, C07D471, C07D487, C07F5, C07F13, C07F15, C07K16