NZ252554A

NZ252554A - Bicyclopolyazamacrocyclophosphonic acid compounds, complexes, conjugates, as contrast imaging agents

Info

Publication number: NZ252554A
Application number: NZ252554A
Authority: NZ
Inventors: Garry E Kiefer; Jaime Simon; Joseph R Garlich
Original assignee: Dow Chemical Co
Priority date: 1993-05-06
Filing date: 1993-05-06
Publication date: 1998-01-26
Also published as: AU665689B2; AU4238293A; WO1994026754A1; NO954442D0; JPH08509976A; NO932823L; FI933507A0; NO954442L; KR950700916A; FI955335A0; KR960702470A; NO932823D0; EP0696290A1; NO304985B1; FI933507A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £52554 New Zealand No. 252554 International No. PCT/US93/04325 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 10.12.1992; Complete Specification Filed: 06.05.1993 Classification:^) C07F9/6561; A61K49/00; A61K47/48; C07F15/02; C07F13/00 Publication date: 26 January 1998 Journal No.: 1424 Title of Invention: Bicyclopolyazamacrocyclophosphonic acids, their complexes and conjugates, for use contrast agents, and processes for their preparation Name, address and nationality of applicant(s) as in international application form: THE DOW CHEMICAL COMPANY, a Delaware corporation of 2030 Dow Center, Abbott Road, Midland, Michigan 48640, United States of America NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION New Zealand No. 252554 International No. PCT/US93/04325 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Bicyclopolyazamacrocyclophosphonic acids, their complexes and conjugates, for use as contrast agents, and processes for their preparation Name, address and nationality of applicant(s) as in international application form: THE DOW CHEMICAL COMPANY, a Delaware corporation of 2030 Dow Center, Abbott Road, Midland, Michigan 48640, United States of America (FOLLOWED BY PAGE 1A) 252554 WO 94/26754 PCT/US93/04325 BICYCLOPOLYAZAMACROCYCLOPHOSPHONIC ACIDS, THEIR COMPLEXES AND CONJUGATES, FOR USE AS CONTRAST AGENTS, AND PROCESSES FOR THEIR PREPARATION This invention concerns ligands that are bicydopolyazamacrocydophosphonic 5 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. To better understand this invention, a brief background on MRI is provided in the following section. Background 10 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. Thistechnique 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 magnetic field. Once 15 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 ryjciei 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 20 which give information on the degree of molecular organization and interaction of protons with the surrounding environment Since the 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 25 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 and that the effectiveness of the chelate in this regard is related, in part, to the number of unpaired 30 electrons producing the magnetic moment [Magnetic Resonance Annua!, 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. It has therefore been proposed that stable, non-toxic 35 paramagnetic chelates be administered to animals in order to increase the diagnostic information obtained by MRI [Frontiers of Biol. Energetics 1752-759(1978); J. Nucl. Med. 25, 506-513 (1984); Proc. of NMR Imaging Svmp. (Oct. 26-27,1980); F. A. Cotton et al.. Adv. Inorg. ■A- WO 94/26754 PCT/US93/04325 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 5 paramagnetic metal ions are gadolinium (Gd*3), iron(Fe°), manganese (Mn*2) and (Mn*J), 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. GdCI3), 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 1 o 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 15 proton NMR relaxation times in the liver or bile duct of an animal using Fe*3-ethylene-bis(2-hydroxy phenyl glycine) complexes and its derivatives, and suggests among various other compounds the possible us^of a pyridine macrocyclomethylenecarboxylicacid. 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 20 gadolinium complexes for MRI which are non-cyclic compounds. C. J. Broan et al., J. Chem. Soc., Chem. Commun., 1739-1741 (1990) describe some Afunctional macrocydic phosphinic acid compounds. C. J. Broan etal.,7. Chem. Soc., Chem. Commun., 1738-1739(1990) describe compounds that are triazabicydo compounds. I. K. Adzamli etal., J. Med. Chem. 32,139-144 (1989) describes acyclic phosphonate derivatives of gadolinium complexes for NMR imaging. 25 Atthe present time, the only commercial contrast agents available in the U.S.A. are the complex of gadolinium with diethylenetriaminepentaacetic acid (DTPA-Gd *3 -MAGNEVIST*" by Schering AG) and a D03A derivative [1l4,7-tris(carboxymethyl)-10-(2-hydroxypropyl)-1,4,7,10-tetraazacydododecanato]gadolinium (PROHANCE™ by Squibb). MAGNEVIST™ and PROHANCE™ are each considered as a non-specific/perfusion agent since it 30 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 macrocydic perfusion agent (DOTAREM'") which presently is only available in 35 Europe. PROHANCE™ is shown to have fewer side effects than Magnevist'". A number of other potential contrast agents are in various stages of development. t Surprisingly, it has now been found that various bicydopolyazamacrocydo-phosphonic acid ligands can be contrast agents. Furthermore, these ligands may have their -2- 38.662B-F 25255* charge modified, i.e. by the structure of the ligand and metal selected, which can effect their ability to be more site specific. Specifically, the present invention is directed to novel ligands that are bicydopolyazamacrocydophosphonic add compounds of the formula Q ^ ^ (I) R-N N-R 10 N I R wherein: 15 R = - f <C)n -T; I Y 20 where: X and Y are independently H, OH, Ct-C3 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 moreH; T is H, C,-C1S alkyl, COOH, OH, SO,H, R2 or -P-OH T 30 where: R' is -0-(C alkyl); R1 is H or OH; with the proviso that when RJ is OH, then the R term containing the R1 must have all X and Y equal to H; R4 is H, NOj, NHj, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl; with the proviso that at least one T must be P(0)R'0H, and with the proviso that when one T is .— R4 -3- OFFf 19 MOV 1997 f^:CsJVEO u 38.662B-F 2 51 □ b ** then one X or Y of that R term may be COOH and all other X and Y terms of that R term must be H; A is CH, N, C-Br, C-d, C-ORJ, C-OR1, N ~-Rl X", 1 OC-C (oy^R4 R3 is H, C(-Cs alkyl, benzyl, or benzyl substituted with at least one R4; R4 is defined as above; Rs is C-C,. alkyl, benzyl, or benzyl substituted with at least one R4; 10 . R* is C,-C1S alkyl ami no; X isCI', Br", I" or HjCCO/; Q and Z independently are CH, N, N*-Rs X\ C-CHrOR3 or C-C(0)-R6; Rs is defined as above; R6 is -0-(C,-C, alkyl), OH or NHR7; 15 7- R is Ct-C5 alkyl or a dextran, a peptide or a molecule with a specific affinity for a receptor; or pharmaceutically-acceptable salts thereof; with the proviso that: a) when Q, A or Z is N or N *-R5X\ then the other two groups must be CH; 20 . b) when A is C-Br, C-Q, C-ORJ or C-OR1, then both Q and Z must be CH; c) the sum of the R4, R7 and R* terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(0)-Rs and when one of Q or Z is C-C(0)-Rs, then A must be CH. 25 30 When the above ligands of Formula (I) have: 35 in the R term three T equal P(0)R'0H, where R' is and n, X, Y, A, Q and Z are defined as above; then the ligands are useful as contrast agents. -0-(C,-C$ alkyl), •4- N-Z. PATENT OFRCF .19 NOV 1997 252554 Particularly preferred are those ligands of Formula (I) where: X and Y are H; nis 1; or A, QandZareCH. 25 35 10 (Afunctional 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 \ ^ ^ OC 15 where R2 and R4 are defined as above, especially where in the two R terms not containing an R4term, both T terms are P(0)R'0H, where R' is defined as above or where in the two R terms not containing an R4 term, one T term is a COOH and the 2o other T term is P(0)R'0H, where R1 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 nis 1 and/or the remaining X and Y terms are H; or A is C-OR3 or C-OR8, where R3 and R8 are defined as above or C-CsC where R4 is defined as above; or A is CH, and one of Q or Z is CH and the other is C-C(0)-R®, where R6 is defined as above; especially those ligands where is NHR^, where R is a dextran, a peptide or a molecule with a specific affinity for a/ receptor. The ligands of Formula (I) may be complexed with various metal ions, such as gadolinium (Gd*3), iron(Fe"5), and manganese (Mn**), 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. ^TTopfice -5- . 9 NOV 1997 'rJiQ 1 38.662B-F 252554 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: 10 15 20 (A)an overall neutral charge • when in the three R terms T is P(0)R'0H, where R* is -0-(C^-Cs alkyl), and n is 1; or (B)an overall charge of + 1 - when one of A, QorZis N*-RSX\ where R'and X"are defined as above; and in oneR term, the T moiety is P(0)R'0H, where R' is -O-^-Cj alkyl); and in the other two R 25 terms, the T moiety is COOH or P(0)R'0H, where R' is -O-^-Cj alkyl); and all X and Y terms are H. Both the complexes and conjugates may be formulated to be in a pharmaceutical^ acceptable form for administration to an animal. Use of the ligands of Formula (I) with other metal ions for diagnosis of disease 30 states such as cancer is possible. The compounds of Formula (I) are numbered for nomenclature purposes as follows: One aspect of the present invention concerns development of contrast agents having synthetic modifications to the paramagnetic chelate enabling site specific delivery of 35 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 aiyllipophilic character of the ^'•^RATE^OFFicF 9 NOV 1997 r ^cbvId" WO 94/26754 PCT/US93/04325 13 10 15 20 25 30 35 (I) complex. The overall range of the charge of the complex is from -3 to +1. For example, for a complex having 2 or more P03H2 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 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. One possible application of this approach is through 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 the contrast agent efficiency resulting in improved contrast relative to the unbound chelate. Recent work by 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 lipophilic character favors non-covalent interactions with blood proteins resulting in enhancement of reiaxivity. 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 th<; invention is made (e.g. possibly -2 or -3 for bone, -1 for liver, or + 1 for heart), the variations n 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) are further defined as follows. "C,-C3 alkyl", "C^C alkyl", "C,-Cl8 alkyl", include both straight and branched chain alkyl groups. An "animal" includes a warmblooded mammal, preferably a human being. -7- ^WO 94/26754 PCT/US93/04325 "Biologically active material" refers to a dextran, peptide, or molecules that havv. 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 5 fragments and F(ab')2 fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes. When usingthe 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 10 (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-19-9,1116-NS-3d, 703D4, 704A1, CC49, CC83 and B72.3 are deposited with the American Type Culture Collection, having the accession numbers ATCC H3 8059, ATCC CRL 8019, ATCC HB 8301, ATCC HB 8302, ATCC HB 9459, ATCC HB 9453 and ATCC HB 8108, respectively. 15 As used herein, "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; "conjugate" refers to a me^l ion chelate that iscovalently attached to an antibody or antibody fragment. The terms "Afunctional coordinator", "Afunctional chelating agent" and "functionalized chelant" are used interchangeably and refer to compounds that have a chelant 20 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 Afunctional 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 25 (represented by R4 or R8 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'. 30 As used herein, "pharmaceutically-acceptable 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. Representative of those salts formed by standard reactions from both organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, 35 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. Also included are salts formed by standard reactions from both organic and inorganic sources such as -8- WO 94/26754 PCT/US93/04325 ammonium or 1-deoxy-1-(methylamino)-l>-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. Detailed Description of the Process The compounds of Formula (I) 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 X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T = P03H2. and Q, A and Z = CH. 15 20 25 30 35 -9- Scheme 1 o « S0C12 t s 80°Cf 2 hours p CI I-II I NaN N NNa I II Ts Ts Ts DMP, 100°C (2) CI HBr/AcOH reflux H3PO3/CH2O HCl NH ——— = ► , N . I S H (4) -TS PO3H2 (5) a compound of Formula (I) WO 94/26754 PCTAJS93/04325 Scheme 2 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T « -Ij'-OH; R1 where R1 = -O-tC^Cj alkyl); and Q, A and Z = CH. Scheme 2 N3 I (4) or i? HP(0Et)2 or p(0Et)3 (4) X-CH2-P(OEt)2 (X » Br, I, CI) CH3CN, k2co3 ? -OHOe^, P (OEt) 2 « ' ^ (EtO)2P "OH{2 eq) O a coumpound of Formula (I) (EtO)2P II O (8) a coumpound of Formula (I) a coumpound of Formula (I) WO 94/26754 PCT/US93/04325 Scheme 3 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T -P-OH; R1 where R1 = Cj-C,. alkyl; and Q, A and Z = CH. Scheme 3 (1) H3CP(OC2H5)2 CH20, THP <2) -0H(3 eq) H-P~CH2CH3 I OH CH20, HCl HO-P /II H3C O HO-P /II H3CH2C o (10) a coumpound of Formula (I) O II P-CH2CH3 (11) P-OH 0^H2CH3 a coumpound of Formula (I) .WO 94/26754 PCT/US93/04325 Scheme 4 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T B -P-OH; where R1 = -O-JC^Cj alkyl) or alkyl; A = C-Br, and Q and Z = CH. -15- Scheme 4 1 2 2 CT» I 11-ESy. 0=^ " 2) EtOH 1 NaBH^ EtOH EtO OEt (13) I—II 1 NaN N NNa I I I Ts Ts Ts » DMP, 100°C SOCI2 (15) HO OH (14) 80°C, 2 hrs. HBr/AcOH Ts- N-TS reClux l Ts (16) 3 a Scheme 4 Cont'd h2o3p Et (1) h3cp(oc2h5)2 ch2o, thf <2) -0H(3 eq) Br a coumpound o Formula (I) h3c ^ N N HO-P 9 II >/P-OH II o Ml H-P-CH2CH3 h3C q O rtf" °0-f B a coumpound of Br Formula (I) CH20, HCl II O Et A p-oh P(OEt)3 EtOH, CH20 h5c (IS) a coumpound of Formula <I) Vr HO-P 11 7 a coumpound of 0 h5c2" f N I /C&s n n—s / (20) >ound Formula (I) p-oh II O P-OH II o WO 94/26754 PCT/US93/04325 Scheme 5 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T -P-OH; ll where R1 = -0-(C,-C5 alkyl) or C,-Cs alkyl; A = C-C-C R4 = H, N0J( NH2 or SCN; and Q and Z = CH. -18- Scheme 5 3 0 E 1 £ vO I Pd(PPh3)2Cl2r (16) Cul, 5 - H Na/Hg amalgam Ts-N N-Ts N . I TS (22) (23) 3 i Ul I I t 2 i to O I 9 (23) HP(OEt)2 EtOH, CH20 (OEt)2P//^N O Scheme 5 Cont'd -0H(3 eq) nA 7 P(OEt)2 ^P(OEt)2 ° (24) P(OEt)3 Et0H,yCH20 0 (23) X-CH2-P(OEt) (X ^ Br, I, CI) CH3CN, K2CO3 (23) (25) a compound of Formula (I) a compound of Formula (I) i Oi i uj l/l 4 * i & K Scheme 5 Cont'd WO 94/26754 PCT/US93/04325 Scheme 6 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T I -P-OH; R1 where R1 = -O-fC^Cj alkyl) or Ct-Cs alkyl; A = C-OR8, where R8 = C,-C5alkylamino; and Q and Z = CH. » -22- WO 94/26754 PCT/US93/04325 VD .c o CO ss o CN n 0) Ul 3 CN O r) £ a o CN CO % u o o r ® O X -23- Scheme 6 Cont'd K> I (35) HBr/AcOH reflux a^H! 1? HP(OEt)2 or P(OEt)3 J EtOH, CH20 MHBOC /W ./W* HBOC W\/HB0C BOC-ON HN > (36) (37) n n^/°h ^TT EtO-P | | P-OEt N'A* II • <0Et,2iT rEt)2 0 ° P(OEt)2 0 "OH(3 eq) II ► II (38) (39) X-CH2~P(OEt)2 X = Br, I, CI) CH3CN 3 o S i ro m I (39) F3CCO2H, ice bath Scheme 6 Cont'd (1) 6N HCl (2) F3CCO2H HO. . ^ \ /\n EtO-P | . XDH n/\ / | P-OEt P<-OEt l|\ (40) £ OH a compound of Formula (I) .✓W™2 /Ng t o N (BO|jt w II <\p-(oh)2 II P—(OH)2 II O (41) a compound of Formula (I) (37) Scheme 6 Cont'd (1) H3CP(OC2H5)2 CH20, THF aa/8' (2) F3CCO2H (3) "OH H-P-CH2CH3 CH20, HCl (2) F3CCO2H n/\ / • P-OH (42) H5C2 HO-P * NH2 a compound of /vN/ Formula {I) ^ yC2H5 n/\ / P-OH II 0 a compound of Formula (I) (43) WO 94/26754 PCT/US93/04325 Scheme 7 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), f fl -P-OH ? R1 where R1 = -OH,-0-(C,-C5 alkyl) or C,-C5 alkyl; Z = C-C(0)-R6, where R* = OH; and Q and A = CH. -27- Scheme 7 Scheme 7 Cont'd I i to vO I H203-P /\n <P- OH ^ hipoi/chao, hcl • p-o3h2 HP(O) (OEt)2rNsCH20, EtOH 0 X-CH2-P (O) (OEt) 2 °H (X » Br%I, CI) (48) CH3CN, K2CO3 " (OEt)^ f^OEt), p-o3h2 h3c \ /\n ho-p " (49) H3CP(0C2H5)2 Jj CH20, THST CH2Of HC3 ^OH 0 <T 0 ^ \p(OEt)2 r" n Xv sCH* n/\ / ' p-oh ^P-OH UXCH3 (50) a compound of Formula {I) h5c2 ^ \ /\n HO-P ' (52) P-OH VC2H5 (51) a compound of Formula (I) . OH n/\/ W P-OEt (53) a compound of Formula (I) I WO 94/26754 PCT/US93/04325 Scheme 8 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T = 0 II -P-OH ; R1 where R1 = -OH, -O-fC^Cj alkyl) or C,-C5 alkyl; Z = C-CHj-OR3 where R3 = benzyl; and QandA = CH. -30- Scheme 8 BzClt CHCI3/K2CO3 (47) _ ► 1 LO Ts- HBr/AcOH reflux Ph Ph HN NH (55) H 0 II H-P-CH2CH3 1 H0 P N " °H CH20/ HCl °H <P- PrOH II O II \ Et <56> a compound of Formula (I) Scheme 8 Cont'd y*°' ► h202-P o Pd/H2 H203P PO3H2 a compound of Formula (I) a compound of Formula (I) Scheme 8 Cont'd (55) i to U3 I 1) H3CP(OC2H5)2 ch2o, thf 2) -OH (3 eq) Pd/H2 (59) a compound of Formula (I) H3C \ A[j HO-P " (60) a compound of Formula (I) Scheme 8 Cont'd O II HP{OEt)2 WO 94/25754 PCT/US93/04325 Scheme 9 prepares the compounds cf Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T 9 -P-OH ; where R* = -OH,-O-tC^Cj alkyl) or C^Cj alkyl; A = N orN-R5; Rs = alkyl halide; and Q and Z = CH. t LO & I ^ N NCS/CCI4, u ?L (H3cco2)2- H3e CH3 * (64) reflux Scheme 9 r*N (X 1 r n . CI CI (65) I—11 I NaN N NNa I I I Ts Ta Ts DMF, 100°C (66) HBr/AcOH reflux I 10 •vj I H2o3p H3P03/CH20, HCl Scheme 9 Cont'd 0 (67) n=l-16 , X=C1, Br, I p03h2 x-(ch2)„-ch3 + > X-N H203P'/Nf N" J po3h2 <PO3H2 1691 a compound of Formula (I) ff ' HP(OEt)2 h-p-ch2ch3 OH CH20, HCl or P(OEt)3 EtOH, CH20 and 2) "0H(3 eq)* N HO-P , H5C2 \ /\n HO-P | II O N ^ xC2li5 ■ P-OH x-ch2-p(oet)2 (X = Br, I, CI) CH3CN, K2CO3 OEt h3cp(oc2h5)2 ch20, thf and 2) 0h(3 eq) ^ p-oh q c2h5 (72) a compound of Formula (I) N H3C \ /\n HO-P " w yCB* n/\ / * P-OH a compound of Formula (I) O <- x p-oh ^XCH3 (71) a compound of Formula (I) 3 o 4* i u> CD I Scheme 9 Cont'd (66) X-(CH2)n-CH3 n=l-16, X=C1, Br, I Ts-N Et0\ A,f HO-P | a compound of OEt (78) Formula (I) w /2H5 " P-OH P(OEt)2 " <p-0H u 0 a compound of!! C2H5 (76) P(OEt)2 Formula (I) J} (77) Cfl 5 WO 94/26754 PCT/US93/04325 Scheme 10 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T if -P-OH ; where R1 = -OH, -O-^-Cj alkyl) or C,-C5 alkyl; Q = N-R5; R5 = CrC,6 alkyl halide; and A and 2 = CH. -39- Scheme 10 I je r" kx NCS/CC1«, (4»-C02)2- r^ N : ► C1 CI r~ir-1 NaN N NNa Ts Ts Ts H3C CH3 (79) reflux DMP,»100°C > Ts-N (80) N-Ts Ts (81) 1 ■e-o ) <X" HBr/AcOH NH •*- (83) X-(CH2)n-CH3 reflux Ts- N-Ts ri=l-16, X=C1, Br, 1 Ts (82) 1 K Scheme 10 Cont'd H,P(WCH,>0, IHCI O II H-P-CH2CH3 po3h2 (88) H203P po3h2 a compound of Formula (I) <X" fffl , hsC2 >°H CH2O, HCl \/^M HO-P • X-CH2~P(OEt)2 (X » Br, I, CI) ch3cn, k2co3 ^ P-OH H3CP(OC2H5)2 0 C2Hs (92) a compound of Formula (I) Eto\ HO-P II 0 N <p- P-OH 11 o "OH(3 eq) a compound of Formula (I) ii'\ OEt (90) a compound of Formula (I) WO 94/26754 PCT/US93/04325 Scheme 11 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T = -P-OH where R1 = -OH, -O-CC,-^ alkyl) or C^Cj alkyl; Q = N or N-R5, Rs = alkyl halide; and A and Z = CH. -42- HBr/AcOH (81) reflux hn nh h (93) 1) h3cp(oc2h5) ch2o, thp Scheme 11 -j>-CH2CH3 oh CH20/ HCl and 2) "OH(3 eq) hsc2 n 1 c2h5 \ /\n / ho-p | i p-oh I' I' O <C 0 x p-oh q c2h5 (95) a compound of Formula (I) ,ch3 h3c \ Am n-Av / ho-p | | p-oh II II o <C o x p-oh j ch3 (94) a compound of Formula (I) Scheme 11 Cont'd (83) 0 1) HP(OEt)2■ EtOH, CH20 or 2) P(EtO)3 H3PO3/CH2O, HCl O or 3) II X-CH2-P(OEt)2 CH3CN, K2CO3 (X - Br, I, Cl) H203P | P(OEt)2 N ^ II P(OEt)2 ° (9g) X-(CH2)n-CH3 N- PO3H2 ^ ' (96) X PO3H2 a compound of Formula (I) H203P a compound of Formula (1)^ "\ HO-P | II >O3h2 ^n^011 OH(3 eq) .OEt PO3H2 (97) OEt <"> a compound of Formula (I) WO 94/26754 PCT/US93/04325 Alternate synthetic procedures allow selective introduction of the phosphonate at the N-6 position. This phosphonate addition is accomplished by the reaction of (4) with formaldheyde sodium bisulfite addition to give quantitative conversion to the 4,9-substituted sulfonate derivative, which is then converted to the corresponding nitrile. Sebsequent phosphonomethylation and hydrolysis yields the desired product. Scheme 12 prepares the compounds of Formula (I) wherein X and Y = H,n s 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), Rat the 3 position has T = -P-OH ; I , whereR1 = -OH or-O-tC^Cj alkyl); and the other two R terms have T = COOH; and A, Qand Z = CH. • Scheme 12 H3PO3/CH2O HCl H203P C02H co2h BrCH2COOH 1 (2 eq) (101) a compound of Formula (I) C02H (100) HP(OEt)2 or P(0Et)3 EtOH, CH20 OH-P EtO^ !! C02H co2h (102) a compound of Formula (I) WO 94/26754 PCT/US93/04325 Scheme 13 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), R at the 3 and 6 positions have T = -P-OH ; where R1 = OH or-0*(C,-C5 alkyl); and the other R term at the 9 position has T = COOH; and A, Q and Z = CH. » t -47- Scheme 13 HCl Na0H/H20 BrCH2COOH (1 eq) H3P03/CH20 - rN r~ n H203P N ^ l co2h po3h2 « HP(OEt)2 or P(OEt)3 EtOH, CH20 2) "OH (106) a compound of Formula (1) (103) OH-P EtO^ H O P-OH (105) Et0/Il a compound of O Formula (I) co2h WO 94/26754 PCT/US93/04325 Scheme 14 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), R terms at the 3 and 9 positions haveT = fl -P-OH ; where R' = -OH or-0-(C,-Cs alkyl); and the other R term at the 6 position hasT = COOH; and A, Qand Z = CH. -49- Scheme 14 EtOH, CH20 l~ N N I H2O3P ► N L co2h p03h2 H07) a compound of Formula (I) 2) "OH OH-P EtO^ !! -N N" I P-OH v/'EtO^J} l C02H (108) a compound of Formula (I) WO 94/26754 PCT/US93/04325 Scheme 15 prepares the compounds of Formula (I) wherein n = 1 (but would also apply if n = 2 or3 with the corresponding change in the reagent), R terms at the 3 and 9 positions have T = ? -P-OH ; where R' = -OH or -O-tC^Cj alkyl); and X and Y = H; the R term at the 6 position has T = I4 9 where R4 = N02 or NH2; and one of X or Y = H and the other = COOH; and A. Q and Z = CH. ♦ -51- WO 94/26754 PCT/US93/04325 -52- Scheme 15 Cont'd j Lri U> (110) hipoi/choo Pt02/H2 HCl /—N P03h2 H2O3P H02C N-^ PO3H2 (HI) a (112) compound of Formula (I) Scheme 15 Cont'd HP(OEt)2 or P(OEt)3 EtOH, CH20 I || 'J 2) "OH ^ N Pt02/H2 -N N' OH-P EtC" I' ^ P-OH Et0^J} OH-P EtO^ II O (113) a compound of Formula (I) (114) a compound of Formula (I) WO 94/26754 PCT/US93/04325 Scheme 16 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), R terms at the 3 and 6 positions have T = ■9 -P-OH ; where R1 = -OH or -0-(C,-C5 alkyl); and X and Y = H; the R term at the 9 position has T = 9 where R4 = NOzor NH2; and one of XorY = H and the other = COOH, A, Q and Z = CH. t -55- Scheme 16 (109) i i_n O* I h3po3/ch2o HCl f H2O3P \ . N co2h h2o3p J NO, (115) a compound of Formula (I) Pt02/H2 H203P (116) a compound of Formula (I) 3 i Scheme 16 Cont'd (117) <118> a compound of Formula (I) a c°n,Pounc* of Formula (I) WO 94/26754 PCT/US93/04325 Scheme 17 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 has T = r -P-OH ; ki where R1 = -OH; and X and Y = H; the Rterm at the 3 and 9 positions have T = COOH; and A, Qand Z = CH. 58 Ol CO Scheme 17 NaCN NH + H0CH2S03Na (121) Lpo3R2 HCl heat/ reflux (120) jsj n 1 m ch20 ' N J C°2H (122) a compound of Formula (I) j pq3h2 ho2C o 2 g 3 § I K WO 94/26754 PCT/US93/04325 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 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 similarity reactive product which would work well in subsequent ring closure reactions. Macrocydization procedures are numerous in the literature and the desired tetraazamacrocyde (3) was prepared according to the method of Stetter et al., Tetrahedron 37, 10 767-772 (1981). More general procedures have since been published which give good yields of similar macrocydes using milder conditions [A. D. Sherry et al., J. Org. Chem. 54,2990-2992 (1989)]. Oetosylation 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. Phosphonomethylation to obtain the tris-aminophosphonic acid derative (5, PCTMP) was conducted under typical Mannich base conditions using phosphorous acid and formaldehyde. In addition to phosphonicacid derivatives, 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 dialkylphosphite as the nudeophilic species (see Scheme 2). Depending upon the reactivity of the amine, these reactions may be conducted at a temperature between about -10 to about 100°C In addition, 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 25 presence of a solvent. When 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 a-halo-dialkylphosphonates in solvents such as acetonitrile, chloroform, dimethylformamide, 30 tetrahydrofuran or 1,4-dioxane with or without the addition of a non-nucleophilic base such as potassium carbonate at room temperature or above. The resulting perester intermediate is then readily hydrolyzed under basic conditions (aqueous hydroxide, pH = 8-14,30-110°C) to give the corresponding half-acid derivative. In Scheme 3, macrocydic methylphosphinic acids (10 and 11) are prepared under conditions similar to those described in Scheme 2. Using diethoxymethylphosphine as the nudeophilic species and paraformaldehyde, condensation can be conducted in solvents such as tetrahydrofuran, dimethylformamide, dioxane, acetonitrile or alcholic media. The resulting -60- WO 94/26754 PCT/US93/04325 phosphinate ester is then hydrolyzed under acid (6N HCl, 80-100°C) or basic (stoichiometric quantities of base, 40-100°Q conditions to give the corresponding methylphosphonic acid. Alternatively, the method devised by A. D. Sherry et al. [Inorg. Chem., submitted 1991) using ethyl phosphonic acid generated in situ can be used to obtain phosphinate derivatives having 5 increased lipophilic character. Scheme 4 illustrates an approach to incorporate additional functionality into the pyridine unit of the 12-memberedtertaazamacrocyde. Thus, chelidamic acid (Sigma Chemical Company; 12) can be converted tothebis-halomethyl derivative (13) having appropriate substitution at the pyridyl 4-position. Transformations leading to this intermediate are general 1 o in nature and its preparation is described by Takalo et al. [Acta Chemica Scandinavica B 42,373-377 (1988)]. Subsequent macrocydization using this intermediate (15) can be accomplished by the standard DMF reaction at 100°C with the sodiotritosylated triamine, or at room temperature with thetritosylated free base and potassium carbonate, sodium carbonate, or cesium carbonate as base to give products similar to those previously described. Subsequent 15 reactions leading to phosphonate half-acids and phosphinate functionality are identical to those transformations and conditions described in the preceeding Schemes. In Scheme 4,4-halopyridyl substituted macrocydes (16) are described which can undergo substitution at the 4-position of the pyridyl moiety as described in Scheme 5. Thus, organometallic Pd(ll) complexes can be employed to facilitate the coupling reaction between 20 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°C for optimum yields. The identical product can also be obtained using Cu(l) phenyl acetyl ide in anhydrous pyridine at a temperature between about 80 to about 110°C. In addition, standard anionic alkylation 25 procedures can be employed to affect substitution on the pyridine nucleus with, for example, sodioalkoxides in DMFordioxaneatfrom about 80 to about 100°C using bases such as potassium carbonate or sodium hydroxide. Macrocydic tetraazamacrocydes (24,25,26,27,28) dervatized in this manner are compatible with transformations described in previous Schemes resulting in analogous phosphonate chelants. 30 A variation of 4-pyridyl substitution is described in Scheme 6 whereby the 4- hydroxypyridyl moiety (29) is alkylated with a bromoalkylnitrile yielding an intermediate ether linked nitrile (31) which is subsequently incorporated into the macrocydic structure. This type of alkylation procedure is best accomplished under anhydrous conditions in an aprotic solvent such as tetrahydrofuran (THF) and using a non-nudeophilic base such as sodium hydride or 35 butyllithium at temperatures between from about -30 to about 80°C. The generality of this approach has been described by Chaubet et al., for acyclic analogs [Tetrahedron Letters 31 (40). ♦ 5729-5732 (1990)]. The macrocydic nitrile prepared in this manner can be reduced to the primary amine (36) by standard procedures followed by protection of the primary amine with -61- WO 94/26754 PCT/US93/04325 2-(t-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON; 37). Subsequent functionalization of the macrocydic secondary amines (38,39,40,41,42,43) can then be accomplished by the procedures discussed with the additional requirement that the BOC protecting group be removed using trifluoroacetic acid as described in Scheme 6. 5 Functionalization can also be carried out on the 3-position of the pyridine ring within the macrocydic structure as illusatrated in Scheme 7. Newkome et al. [Tetrahedron 39(12). 2001-2008 (1983)1 has previously described the synthesis of ethyl 2,6-halomethylnicotinate (45) which serves as the inital starting material in this synthetic route. Thus, the tris-tosylated macrocyde intermediate (46) can be detos/lated under acidic 10 conditions (HBr/AcOH, 25-115°C) with simultaneous hydrolysis to yield the nicotinic acid derivative (48), or reduction of the ester in refluxing ethanol priorto detosylation will result in the 3-hydroxymethyl intermediate (47). The nicotinic acid macrocyde 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). 15 In contrast, the 3-hydroxymethyl analog is advantageously protected prior to functionalization of the macrocydic 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 20 catalytic hydrogenation conditions (58). Macrocydic derivatives can also be prepared as in Schemes 12-14 where both carboxylate and phosphonate chelating fuctionalities are present in the same molecule. Thus, varying degrees of carboxylatefuctionality can be introduced under typical aqueous alkylation procedures using bromoaceticacid. Following this step, the remaining amines can be phos-25 phonomethylated 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 substituted at one of the macrocydic nitrogen positions. Typically, the macrocydic amine is mono-N-functionalized in an organic solvent such as acetonitrile or DMF at room 30 temperature using a non-nudeophilic base such as potassium carbonate. Additional functionalization of the remaining nitrogen positions is then performed by methodsand 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 35 to larger synthetic ot natural molecules. Scheme 17 illustrates the synthesis of the macrocydic 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 -62- WO 94/26754 PCT/US93/04325 macrocydic compound where positions 3 and 9 are the sodium salt of methanesuifonic 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 5 sequential reaction with a derivative of phosphorous acid and formaldehyde to form the phosphonic acid at the 6 position (121), followed by add hydrolysis, atan elevated temperature, of the cyanato groups and any derivative moiety of the phosphorous acid present The resulting compound is a macrocyde with two carboxylic acid groups at positions 3 and 9 and a phosphonic acid group at position 6. The phosphonomethylation can also be 10 preformed by the methods discussed above. The metal ions used to form the complexes of this invention are Gd*\ Mn*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 -ramagnetic nuclide" of this invention means a metal ion which displays spin 15 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*J), with Gd*3 being preferred. 20 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 bicydopoiyazamacrocydophosphonic acid with 25 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. 30 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 35 for example, Kohlerand Milstein fNature. 256.495-497 (1975): and Eur. J. Immunol., 6,511-519 (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 inthe conjugates are monoclonal antibodies, or fragments -63- WO 94/26754 PCT/US93/04325 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. Sone examples of 5 antibodies or antibody fragraments are 1116-NS-19-9,11l6-NS-3d, 703D4,704A1.CC49, CC83 and B723. 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 1 g 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, maybe used. An "effective amount" of the formulation is used for diagnosis. The dose will 15 vary depending on the disease and physical parameters of the animal, such as weight In vivo diagnosticsarealsocorrtemplated using formulations of this invention. Other uses of some of the cheiants 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. 2Q The ligands of Formula (I) having in at least two R terms T equal to P(0)R'0H 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. The invention will be further clarified by a consideration of the following 25 examples, which are intended to be purely exemplary of the present invention. Some terms us^d in the following examples are defined as follows: LC = liquid chromatography, purifications were carried out at low pressure using Dionex2010i system fitted with a hand-packed Q-Sepharose™ anion exchange column (23 x 2 cm). 30 DMF = dimethylforamide. AcOH = acetic acid. ICP = inductively coupled plasma. g = gram(s). mg = milligrams. 35 kg = kilogram(s). mL = milliliters). pL = microliters). -64- ^\v O 94/26754 PCT/US93/04325 dH Stability General Procedure A stock ""GdCI, (or ,53SmCI3) solution was prepared by adding 2 yX. of 3x1 CM "9GdQ} in 0.1N HO to 2 mLof a 3x1 CM GdCI3 carrier solution. Appropriate ligand solutions were then prepared in deionized water. The 1:1 ligand/metal complexes were then prepared 5 bycombinfng the ligands (dissolved In 100-500 i*L of deionized water) with 2 mL of the stock "®GdCI3 solution, followed by through mixing to give an acidfc solution (pH = 2). The pH of the solution was then raised to 7.0 using 0.1N NaOH. The percent metal as a complex was then determined by passing a sample of the complex solution through a Sephadex" G-50 column, eluting with 4: f saline (85% Naa/NH40H) and collecting 2 x 3 mL fractions. The amount of 10 radioactivity in the combined elutiohs was then compared with that left on the resin (non-com piexed metal is retained on the resin). The pK stability profile was generated by adjusting the pH of an aliquot of the complex solution using 1M NaOH or 1M HQ 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 15 biodistribution of the ligands of this invention. STARTING MATERrALS Example A Preparation of 2,6-bis(chloromethy!)pyridine. To 100 mL of thionyl chloride that was cooled (ice bath) was added 24 g (0.17 mol) 20 of 2,6-bis(hydroxymethyl)pyridine. After 30 min, the reaction mixture was warmed to room, temperature, then refluxed for 1.5 hi?. After cooling the reaction mixture to room temperature, the solid which formed was filtered, washed with benzene and dried in vacuo. The solid was then neutralized with saturated NaHC03, filtered and dried to yield 23.1 g (71.5%) of the titled product as an off-white crystalline solid, mp 74.5-75.5°C, and further 25 characterized by: 'H NMR(CDCip 8 4.88 (S, 4H), 7.25-7.95 (m. 3H). Example B Preparation of 3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicydo[9.3.1 ]pentadeca-1(15), 11,13-30' triene. A DMF solution (92 mL) of 6.9 g (11.4mmol) of 1,4,7-tris(p-tolylsuifonyl)diethylenetriaminedisodium salt was stirred and heated to 100°C under nitrogen. To the solution was added dropwise over 45 min 2 g (11.4 mmoJ) of 2,6-bis(chloromethyl)pyridine (prepared by the procedure of Example A) in 37 mLof DMF. When 35 the addition was completed the reaaion mixture was stirred at 40°C for 12 hrs. To the reaction mixture was then added 50-75 mL of water, resulting in immediate dissolution of NaCI, followed by precipitation of the title product. The resulti ng slurry was then filtered and the solid washed with water and dried in vacuo. The title product was obtained as a light-tan powder, 6.5 g (86%), mp 168-170°C dec. and further characterized by: -65- WO 94126754 PCT/US93/04325 'H NMR (CDCIj) 5 2.40 (s, 3H), 2.44 (s, 6H), 2.75 (m, 4H), 3.30 (m, 4H), 4.28 (s, 4H), 7.27 (d, 2H), 7.34 (d, 4H), 7.43 (d, 2H), 7.65 (d, 4H), 7.75 (t, 1H); and ,JCNMR 5 6 21.48,47.29,50.37,54.86,124.19,127.00,127.11,129.73,135.04,135.74,138.95,143.42, 143.73,155.15. Example C Preparation of 3,6,9,15-tetraazabicyclo(9.3.1 Jpentadeca-1 (15), 11,13-triene. A solution of HBr and AcOH was prepared by mixing 48% HBr and glacial AcOH in a 64:35 ratio. To 112 mL of the HBr/AcOH mixture was added 5.5 g (8.2 mmol) of 3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicydo[9.3.1 Jpentadeca-1 (15), 11,13-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 15 vigorously and 15-20 mLof diethyl ether was added. A off-white solid formed which was filtered, washed with diethyl ether, and dried in vacuo. The dry tetrahydrobromide salt was then dissolved in 10 mL of water, adjusted to pH 9.5 with NaOH (50% w/w) and continuously extracted with chloroform for 4 hrs. After drying over anhydrous sodium sulfate, the chloroform was evaporated to give a ligh -tan oil which gradually crystallized upon standing at 20 room temperature to yield 1.2g(71%)ofthetitleproduct,mp86-88°Candfurther characterized by: 'H NMR (CDCI3) 8 2.21 (m, 4H), 2.59 (m, 4H), 3.06 (s, 3H), 3.85 (s, 4H), 6.89 (d, 2H), 7.44 (t, 1H); and ,3C NMR 25 8 48.73,49.01,53.63,119.67,136.29,159.54. Example D Preparation of 3,6,9,15-tetraazabicyclo[9.3.1 Jpentadeca-1 (15), 11,13-triene-3,9-dimethylenesulfonic acid. A slurry of 500 mg (2.4 mmol) of 3,6,9,15-tetraazabityclo[9.3.1]pentadeca-30 1(15),11,13-triene (prepared bythe procedure of Example C) was stirred in 6mLof water and the pH adjusted to 3 using 6M HQ. To the mixture was added 682 mg (5.1 mmol) of hydroxymathanesulfonic acid sodium salt and the pH adjusted to 9 with 50% aqueous sodium hydroxide. After stirring for three hrs at room temperature, "C NMR indicated complete conversion to the title bis-methylenesulfonic acid product. 35 -66- WO 94/26754 PCT/US93/04325 Example E Preparation of 3,6,9,15-tetraazabicydo[9.3.1 lpentadeca-1 (15), 11,13-triene-3,9-dimethylenenitrile. Tothe reaction mixture containing 3,6,9,15-tetraazabicydo(9.3.1]pentadeca-5 1(15),11,13-triene-3,9-dimethylenesulfonic acid from Example D was added 47 mg (9.6 mmol) of sodium cyanide. The reaction mixture was stirred at room temperature for 24 hrs. ,3C NMR indicated that transformation to the bis-nitrile was complete. The reaction mixture was then filtered, extracted three x 25 mL with chloroform, dried over anhydrous magnesium sulfate, and concentrated to give a viscous oil. The oil was then disolved in chloroform, triturated with 10 cydohexane, and concentrated to give, as white powder, 530 mg (78%) of the title dimethylenenitrile product. Example F Preparation of 3,9-bis(sodium methylenesulfonate)-3,6,9,15-tetraazabicydo[9.3.Upentadeca-1(l5),11,13-triene (PC2S). 15 An aqueous solution (10.0 mL) of 3,6,9,15-tetraazabicyclo[9.3.1 jpentadeca- 1(15), 11,13-triene (prepared by the procedure of Example C), 1.03 g (5.0 mmol) was added with 0.5 mL of concentrated HCl and stirred for 10 min to ensure complete dissolution. The resulting solution had a pH of 8.6. Tothe solution was then added 1.37 g (10.2 mmol) of H0CH2S03Na with 5 mL of deionized water. The solution was heated at 60°C for 10 min and the pH dropped 20 to 5.6. After cooling, the pH was adjusted to 9.0 with 1M aqueous sodium hydroxide, followed by lyophilization to give the desired product as a white solid in a quantative yield and characterized by: 'H NMR (DjO) 5 2.87 (t, 4H), 3.18 (t, 4H), 3.85 (s, 4H), 4.11 (S, 4H), 7.03 (d, 2H), 7.55 (t, 1H); and 25 ,3CNMR(D20) 848.52, 54.04,58.92,79.09.123.90, 141.37,161.89. Example G Preparation of 3.9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclo[9.3.1lpentadeca-1(15),11,13-triene. 30 To an aqueous solution, 10.0mL, of 3,9-bis(sodiummethylenesulfonate)-3,6,9,15- tetraazabicydo[9.3.1 Jpentadeca-1 (15), 11,13-triene (prepared by the procedure of Example F), 2.26 g (5 mmol), was added 0.6 g (12.24 mmol) of sodium cyanide. The mixture was stirred for 3 hrs at room temperature. The pH of the reaction mixture was about 10. The pH was adjusted to above 13 with concentrated aqueous sodium hydroxide. The product precipitated and was 35 extracted with chloroform (3 x 20 mL), dried over anhydrous magnesium sulfate, and filtered. Upon removal of solvent and concentration in vacuo, the desired product was isolated as a waxy, white powder, 1.0 g (71 %) and characterized by: -67- WO 94/26754 PCT/US93/04325 'h nmr (cdcij) 6 2.03 (br s, 4H), 2.64 (m, 4H), 3.82 (s, 4H), 3.90 (s, 4H), 7.14 (d, 2H), 7.62 (t, 1H); and ,3cnmr(cdci3) 846.08,46.64,52.89, 60.78,115.31,122.02, 137.57, 157.33. 5 Example H Preparation of 3,9-bis(methylenenitrile)-6-(methylenedimethylphosphonate)-3,6,9,15-tetraazabicydo[9.3.1]pentadeca-1(15),11,13-triene-3,9-dimethylenenitrile. 3,9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (prepared by the procedure of Example G), 285 mg (1.0 mmol) was combined with 60 mg 10 (2.0 mmol, excess) of paraformaldehyde and 0.354 mL (372 mg, 3.0 mmol, excess) of trimethylphosphite. The mixture was gently "itirred for 10 min to obtain a slurry, then heated to 90°C for 1 hr. After the excess reagents and byproducts were removed in vacuo (1 hr at 125°C/0.01 mmHg), he resulting dark brown residue was dissolved in 20 mL of chloroform and washed with deionized water (5x15 mL). The organic layer was dried over anhydrous 15 magnesium sulfate, filtered, and the excess solvents evaporated in vacuo to give the desired product as a yellow waxy solid, 168 mg (41%) and characterized by: 'HNMR(CDCI3) 8 2.61 (brs, 8H), 2.73 (d, 2H), 3.62 and 3.68 (s, 6H), 3.73 (s, 4H), 3.84 (s, 4H), 7.06 (d, 2H), 7.57 (t, 1H);and 20 ,3cnmr(cdci3) 844.44, 50.74,51.03,51.85,52.51,60.28,115.61, 122.27,137.24,156.61. Example I Preparation of 3,6,9,15-tetraazabicydo[9.3.1 Jpentadeca-1 (15), 11,13-triene-3,6,9-methy I ened i ethyl phosphonate. 25 A mixture of 1 g (4.8 mmol) of 3,6,9,15-tetraazabicydo[9.3.1 Jpentadeca- 1(15), 11,13-triene (prepared by the procedure of Example Q, 4.8 g (28.8 mmol) of triethyl phosphite and 864 mg (28.8 mmol) of paraformaldehyde was heated at 90°C with constant stirring for 45 min. The reaction mixture was concentrated in vacuo and the viscous oil chromatographed on a basic alumina column, eluting with chloroform. After concentration of 30 the organic eluent, the desired product was isolated as a colorless oil, 2.0 g (64%) and characterized by: 'HNMRfCDCg 8 1.23 (m, 18H), 2.77 (m, 12H), 3.04 (d, 6H), 4.13 (m, 12H), 7.17 (d, 2H), 7.60 (t, 1H); and ,3c nmr (cdci3) 35 8 16.43, 50.03, 50.31, 50.43,50.77, 51.23,51.38, 52.63, 53.30,60.86,60.92, 61.63,61.74, 61.83, 61.93,62.32,76.46,76.97,77.18,77.48,122.50, 137.10,157.18; and 3,pnmr 8 24.92 (s,2P), 24.97 (s,1P). -68- WO 94/26754 PCT/US93/04325 Example J Preparation of 3,6,9,15-tetraazabicydo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylenedi(n-propyl)phosphonate. To 3 mLof a chloroform/dioxane solution (1:1) was added 100 mg (0.48 mmol) of 5 3,6,9,15-tetraazabicydo[9.3.1lpentadeca-1(15), 11,13-triene (prepared by the procedure of 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, eiuting with chloroform. After concentration 10 of the organic eluent, the desired product was isolated asa colorless oil, 320 mg (90%) and characterized by: 'H NMR(CDCI3) 6 0.88 (m, 18H), 1.61 (m, 12H), 2.72 (m, 12H), 3.03 (d, 6H), 3.97 (m, 12H), 7.13 (d, 2H), 7.55 (t, 1H); and 15 13CNMR (CDCIj) 89.96,23.73,49.84, 50.14, 50.26, 50.57,51.11,51.23, 52.43, 53.01,60.78,60.84,67.27, 67.40, 122.48,137.04,157.16; and » 31PNMR 8 24.98 (3P). 20 Example K Preparation of 3,6,9,15-tetraazabicydo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methy I ened i (n-butyl)phosphonate. A mixture of 500 mg (2.4 mmol) of 3,6,9,15-tetraazabicydo!9.3.1]pentadeca-1(15),11,13-triene (prepared by the procedure of Example C), 2.0 g (8 mmol) of tributyl 25 phosphite and 240 mg (8 mmol) of paraformaldehyde was heated at 100#C with stirring for 1 hr. The resulting viscous solution was concentrated in vacuo to give an oil which was chromatographed on a basic alumina column, eiuting with chloroform. After concentration of the organic eluent, the desired product was isolated as a colorless oil, 1.25 g (65%) and characterized by: 30 'H NMR (CDCIj) 8 0.84(m, 18H), 1.27(m, 12H), 1.58(m, 12H),2.57(>.n, 12H), 3.01 (d,6H), 3.99(m, 12H), 7.12(d, 2H), 7.54 (t, 1H); and 13C NMR (CDCI3) 8 13.42, 13.46, 18.50,18.59, 32.16, 32.43,49.88, 50.03, 50.16, 50.63, 51.11,51.27, 52.48, 53.16, 35 60.71,60.78,65.38,65.48,65.58,122.46,136.96,157.14; and 3,PNMR 8 24.88 (2P), 24.93 (1 P). -69- WO 94/26754 PCT/US93/04325 Example L Preparation of 3,6,9,15-tetraazabicyclo[9.3.1 ]pentadeca-1 (15), 11,13-triene-3[(4-nitrophenyOmethyl acetate]. To a solution of 2.5 mLof chloroform which was rapidly stirred and 200 mg 5 (0.97 mmol) of 3,6,9,15-tetraazabicydo[9.3.1 Jpentadeca-1(15), 11,13-triene (prepared by the procedure of Example C), was added in one portion 266 mg (0.97 mmol) of bromo(4-nitrophenyl)methyl acetate in 2.5 mL of chloroform. The reaction mixture was stirred for 24 hrs at room temperature. The solution was concentrated in vacuo to give a semi-solid which was chromatographed on a silica gel column, eiuting with chloroform/methanol/ammonium 10 hydroxide (16:4:1). After concentration ofthe organic eluent, the desired product was isolated as a light yellow solid, 250 mg (64%) and characterized by: 13C NMR(CDCI3) 8 45.67,45.90,45.97, 51.65,52^08,52.28,53.78,69.54,119.03, 119.23,122.85,130.30,137.06. 143.27,147.05,159.59, 160.41,171.70. 15 FINAL PRODUCTS Example 1 Preparation of 3,6,9,15-tetraazabicydo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-trimethylenephosphonic acid (PCTMP). A mixture of 2.06 g (10 mmol) of 3,6,9,15-tetraazabicydo[9.3.1]pentadeca-20 1 (15), 11,13-triene (prepared by the procedure of Example C), 11.3 g (138 mmol) of phosphoric acid and 15 g (152 mmol) of concentrated HCl was heated to gentle reflux (103 °C) with constant stirring followed by the dropwise addition (2 mL/min) of 12.2 g (150 mmol, 15 mL) of aqueous formaldehyde (37%). After complete addition, the reaction mixture was stirred at reflux for 16 hrs, cooled to room temperature and concentrated to a thick, viscous oil. The 25 pr oduct was then purified by LC anion exchange chromatography (0-30% formic acid, 3 mL/min, retention time = 32 min). The combined fractions were freeze-dried to give 4.8 g (99%) of the title product as a white solid, mp 275-280°Cand further characterized by: 1HNMR(D20) 8 2.83 (m, 6H), 3.46 (m, 10H), 7.28 (d, 2H), 7.78 (t, 1H); and 30 ,3CNMR 8 53.61,53.81,55.27,57.93,62.20,125.48,143.08,152.31; and 31PNMR 8 8.12 (2P), 19.81 (1P). Example 2 35 Preparation of the complex of 153Sm-3,6,9,15-tetraazabicyclo[9.3.1 ]pentadeca-1 (15),11,13-triene-3,6,9-trimethylenephosphonic acid ('S3Sm-PCTMP). A solution ofthe ligand of Example 1 was prepared by dissolving 3.8 mg of ligand/0.517 mLof deionized water (pH = 2). A 1:1 ligand/metal complex was then prepared by -70- WO 94/26754 PCT/US93/04325 combining 40 pi ofthe ligand solution with 2 mL of aqueous SmCljHjO (3x10"*M in 0.01 N HCl) containing tracer 153SmClr After thorough mixing, the percent metal as a complex was determined by passing a sample ofthe complex solution through a Sephadex"* column, eiuting with 4:1 saline (0.85% NaCI/NH40H), and collecting 2x3 mL fractions. The amount of 5 radioactivity in the combined elutions was then compared with that left on the resin. Under these conditions, complex was removed with the eluent and non-complexed metal is retained on the resin. Bythis method complexation was determined to be 98%. A sample of the solution that was passed through the resin was used for pH studies. The pH stability was then determined using the General Procedure above. 10 Example 3 Preparation of 3,9-diacetic acid-6-(methylenephosphonic acid)-3,6,9,15-tetraazabicydo[9.3.1 ]pentadeca-1 (15),11,13-triene (PC2A1P). A concentrated hydrocholric acid solution (37%, 5 mL) of 3,9-bis(methylene-nitrile)-6-(methylenedimethylphosphonate)-3,6,9,15-tetraazabicydo[9.3.1]pentadeca-15 1(15),11,13-triene (prepared in Example H), 168 mg (1.0 mmol) was heated at reflux for 16 hrs. After cooling, the solution was evaporated to dryness, followed by coevaporation with deionized water (2 x 10 mL)£o remove the excess hydrochloric acid. The filal product was isolated as a dark brown solid upon lyphilization of the concentrated queous solution and characterized by: 20 'H NMR(D20) 5 2.68 (br s, 4H), 3.31 (br s, 4H), 4.08 (s. 4H), 4.55 (s,4H), 7.16 (d, 2H), 7.68 (t, 1H); and ,3CNMR(D20) 852.35, 54.04, 57.02, 59.24,62.26, 125.52,143.64,152.36,171.54; and 3'PNMR(D20) 25 820.03. Example 4 Preparation of 3,6,9,15-tetraazabicydo[9.3.1 Jpentadeca-1 (15), 11,13-triene-3,6,9-methyleneethylphosphonate tris(potassium salt) (PMEHE). To an aqueous 0.1N potassium hydroxide solution (2 mL) was added 250 mg 30 (0.38 mmol) of 3,6,9,15-tetraazabicydo[9.3.1 ]pentadeca-1 (15),11,13-triene-3,6,9-methylene-diethylphosphonate (prepared by the procedure of Example I). The solution was heated at 90°C for 5 hrs. The reaction mixture was cooled to room temperature, filtered, and freeze-dried to yield the desired product as an off-white solid, 252 mg (97%) and characterized by: "C NMR (DjO) 35 8 18.98,19.82,51.78, 52.06,53.08, 54.46, 54.68, 57.01, 58.22,60.24,63.19,63.25, 63.36, 63.49, 63.59,63.95,64.18,64.25, 66.80,126.62,141.63,159.40; and 31PNMR 8 20.58 (s, 2P), 20.78 (s, 1P). -71- WO 94/26754 PCT/US93/04325 Example 5 Preparation of 3,6,9,15-tetraazabicydo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylene(n-propyDphosphonate tris(potassium salt) (PMPHE). To an aqueous solution of potassium hydroxide (0.5 mLof 1 N/dioxane (0.5 mL) 5 was added 81 mg(0.108mmol)of3,6,9,l5-tetraazabicydo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylenedi(n-propyl)phosphate (prepared by the procedure of Example J). The solution was heated at reflux for 24 hrs. The reaction mixture was cooled to room temperature and extracted with diethyl ether. The ether extract was then concentrated in vacuo to yield the desired product as an off-white solid, 48.6 mg (60%) and characterized by: 10 3,PNMR 8 20.49 (s, 3P). Example 6 Preparation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylene(n-butyl)phosphonate tris(potassium salt) (PMBHE). 15 To an aqueous solution of 35 mL of 1N potassium hydroxide was added 3.21 g (3.88 mmol) of 3,6,9,15-tetraazabicydo[9-3.1]pentadeca-1(15),11,13-triene-3,6,9-methylenedi(n-butyl)phosphate (prepared by the procedure of Example K). The solution was heated at reflux for 5 days. The reaction mixture was cooled to room temperature, filtered and the filtrate freeze-dried to give a cream colored solid. The solid was then suspensed in 150 mL 20 of methanol and stirred for 12 hrs at room temperature. The slurry was then filtered and the filtrate concentrated to give a semi-solid. The solid was taken up in 150 mL of chloroform and dried over anhydrous sodium sulfate and filtered. After concentration in vacuo the product was isolated as an off-white solid, 1.86 g (62%) and characterized by: 'H NMR (D20) 25 8 0.68 (m, 9H), 1.14 (m, 6H), 1.37 (m, 6H), 2.76 (d, 6H), 3.41 (m, 12H), 3.73 (m, 6H), 7.24 (d, 2H), 7.76 (t, 1H); and ,3CNMR(D20) 8 15.76, 15.80,21.12,21.20,34.96,35.06,35.14, 52.08,52.53,53.38,53.48, 54.49,54.75, 57.70, 57.76,61.86, 67.65, 67.75,67.98,68.08,125.15,142.93,152.25; and 30 3,PNMR 89.73 (s,2P), 21.00 (s,1P). Example 7 Preparation of 3,6,9,15-tetraazabicydo[9.3.1]pentadeca-1(l5),11,13-triene-3[(4-nitrophenyl)methyl acetate]-6,9-methylenediethylphosphonate. 35 A solution of 250 mg (0.62 mmol} of 3,6,9,15-tetraazabicydo[9.3. Upentadeca- 1(15), 11,13-triene-3[(4-nitrophenyl)methyl acetate] (prepared by the procedure of Example L), 624 mg (3.7 mmol) of triethyl phosphite, and 111 mg (3.7 mmol) of paraformaldehyde was stirred at 100°C for 1 hr. The reesulting homogeneous solution was concentrated in vacuo to -72- WO 94/26754 PCT/US93/04325 give a viscous oil. The oil was dissolved in 10 mL of chloroform and washed with water (3x5 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered and the filtrate concentrated in vacuo to give the product as aviscous oil, 326 mg (96%) and characterized by: 3,PNMR(CDCip 5 8 24.67 (s,2P), 24.88 (s, 1P). BIODISTRIBUTION General Procedure Sprague Dawley rats were allowed to acclimate for five days then injected with 10 100 pL ofthe 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 100pL standards in order to determine the percentage of the dose in each tissue or organ. 15 The percentdose in blood was estimated assuming Wood to be 7% ofthe body weight. The percent dose in bone was estimated by multiplying the percent dose in the femur by 25. The percentdose in rfiusde was estimated assuming muscle to be 43% ofthe body weight. in addition to organ biodistribution, chelates of the compounds of Formula (I) 20 were evaluated for efficiency of bone localization since phosphonates are known fortheir ability to bind to hydroxyapatite. EXAMPLE I The percent ofthe injected dose of complex of of Example 2 (153Sm-PCTMP) in several tissues are given in Table I. The numbers represent the average of a minimum of 3 rats 25 per data point at 2 hours post injection. TABLE I % INJECTED DOSE IN SEVERAL TISSUES FOR l53Sm-PCTMP 30 35 TISSUE AVERAGE Bone 34,87 Liver 0.99 Kidney 1.42 Spleen 0.07 Muscle 4.77 Blood 6.27 -73- WO 94/26754 PCT/US93/04325 EXAMPLE II The percent ofthe injected dose of complex of of Example 5 (,53Sm-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. TABLE II % INJECTED DOSE i53Sm-PMPHE (2 hours) TISSUE AVERAGE Bone 10.86 Liver 4.14 Kidney 1.55 Spleen 0.05 Muscle 1.19 Blood 0.25 Heart 0.08 Lung b 0.12 Brain 0.00 Stomach 0.44 Small Intestine 10.71 Large Intestine 2.17 EXAMPLE III 25 The percent ofthe injected dose of complex of of Example 6 (1S3Sm-PMBHE) in several tissues are given in Table III. The numbers represent the average of a minimum of 3 rats per data point at 2 hours post injection. 30 35 -74- WO 94/26754 PCT/US93/04325 TABLE III % INJECTED DOSE l53Sm-PMBHE (2 hours) TISSUE AVERAGE Bone 3.73 Liver 2.70 Kidney 0.43 Spleen 0.05 Muscle 1.09 Blood 0.14 Heart 0.02 Lung 0.04 Brain 0.00 Stomach 0.08 Small Intestine 57.89 Large Intestine 0.77 EXAMPLE IV The percent ofthe injected dose of complex of of Example 3 (153Sm-PC2A1) in several tissues are given in Table IV. The numbers represent the average of a minimum of 3 rats per data point at 2 hours post injection. 25 30 35 -75- </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WO 94/26754 PCT/DS93/04325 TABLE IV % INJECTED DOSE i53Sm-PC2A1P(2 hours) 10 15 IMAGING EXPERIMENTS General Procedure 20 Injectable solutions were first prepared (0.5M) by dissolving the appropriate amount of each complex in 2 mLof 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. An anesthetized Sprague Dawley rat was injected intramuscularly with one of 25 the metal solutions described above at a dose of 0.05-0.1 mmol Gd/kg body weight. Images were then taken at various time intervals and compared witha non-injected control at time 0. Example II The Gd-PCTMP complex (prepared in Example 2) showed kidney enhancement and bone localization in the shoulder, spine and sternum. 30 Other embodiments ofthe invention will be apparent to those skilled in the art from a consideration of this specification or practice ofthe 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. 35 TISSUE AVERAGE Bone 47.98 Liver

1.46

Kidney

0.93

Spleen

0.02

Muscle

1.00

Blood

0.36

Heart

0.04

Lung

0.06

Brain

0.01

Stomach

0.25

Small Intestine

13.10

Large Intestine

0.12

-76-

77

ACLAIMS: _

95^ 55 a

1 . Bicyclopolyazamacrocyclophosphonic acid compounds£jo«r tfce ~

formula o^'sz

-ks 1

R-N N-R

i h j

(I)

wherein:

f

R = -(C)„ -T;

I

Y

where:

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

n is an integer of- 1 , 2 or 3;

'A •• •'

with the proviso "hat: when n is 2, then th*£ sum of X and Y must equal two or more H; ana when n is 3, then ^he sum of X and Y must equal three or more H;

T is H, C,-C,s alkyl, COOH, OH, SOjH,

R2-

R1

where:

Rl is -0-(Ca-C5 alkyl);

R2" is H or OH; with the proviso that when Rs is OK, then the R term containing the R? must have all X and Y equal tp__H

N.Z. PATENT OFFICE

Z 7 NOV mt

25255 4

R4 is H, NOj, NHx, isothiocyanato, semicarbazido,

thiosemicarbazido, maleimido, bromoacetamido or carboxyl;

with the proviso that at least one T must be P(0)R10H and with the proviso that when one T is i— r4

then one X or Y of that R term may be COOH and all other X and' Y terms of that R term must be H;

A is CH, N, C-Br, C-Cl, C-OR"\ C-OR®, N*-R5X~,

C-CsC (qW ;

R3 is H, C^-Cf alkyl, benzyl, or benzyl substituted with at least one R4;

R5 is Cj-Ck alkyl, benzyl, or benzyl substituted with at least one R\*

R6 is Ci-Ci* alkylaminc;

X" is CI", Br", I" or HjCCQ^";

Q and Z independently are CH, N, N+-R5X", C-CH2-OR3 or C-C(0)-R°, R°' is -0-(Ci-C3 alkyl), OH or NHR7;

R7 is Cj-C£ alkyl or a dextran, a peptide or a molecule with a specific affinity for a receptor; or pharmaceutically acceptable salts thereof;

with the proviso that:

a) when Q, A or Z is N or N' -R5X~, then the .nther two

groups must be CH;

n.z. patent office

2 7 NOV 199?

79

25255 4

b) when A is C-Br, C--C1, C-OR3 or C-OR8, then both Q and Z must be CH;

c) the sum of the R4, R7 and R9 terms, when present, may not exceed one; and d) only one of Q or Z can be C-C (0) -R* and when one of Q or z is C-C(0)-R^, then A must be CH.

2. A compound of Claim 1 wherein X and Y are H.

3. A compound of Claim 1 or Claim 2 wherein n is 1.

4. A compound of any one of Claims 1 to 3 wherein A, Q and Z are CH.

5. A compound of any one of the preceding claims wherein in the three R terms T is P(0)Rl0H, where Rl is as defined in Claim 1.

6. A compound of Claim 5 wherein n is 1.

7. A compound of any one of Claims 1 to 4 wherein Q, A and Z are CH; and in the three R terms X, Y and n are defined as in Claim

1, and one T term is are defined as in Claim 1.

8. A compound of Claim 7 wherein n is 1 and the R term that contains a T moiety which has the R4 group present, also has one of X or Y of that R term equal to COOH.

9. A compound of Claim 8 wherein in the two R terms not containing an R^" term, all remaining X and Y terms are H.

or where a?" and R^ are defined in Claim 1 , and the other two T terms n.2. patent office

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10. A compound of any one of Claims 1 to 4 wherein n is 1, X and Y are H; T is COOH

where: R4 is -0-(C^-C^ alkyl) .

11. A compound of claim 10 wherein

(a) Q and Z are CH, A is C-OR3, C-OR8, where R3 and R8 are defined as in Claim 1, or where R4 is defined as in Claim 1 or

(b) A is CH, and one of Q or Z is CH and the other is C-C(0)-R*, where R6 is defined as in Claim 1. .

is a dextran, a peptide, or a molecule with a specific affinity for a receptor.

13. A compound of Claim 4 wherein one of A, Q or Z is N"-R5X", where R5 and X" are defined as in Claim 1; and in all three R terms, the T mcxety is ?(O)R40H, where R1 is -0-(C:-Cj alkyl) and all X and Y terms are H.

14. A complex which comprises a bicyclopolyazamacrocyclophosphonic acid compound as claimed in any one of Claims 1 to 13 complexed with a metal ion selected from Gd*3, Mn*z or Fe43.

if or

12. A compound of type (b) Claim 11 wherein R6 is NHR7, where R'

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A complex of Claim 14 wherein the metal is Gd+3.

16. A complex of Claim 14 having an overall negative charge.

17. A complex of Claim 14 having an overall neutral charge.

18. A complex of Claim 14 having an overall charge of +1.

19. A conjugate comprising a bicyclopolyazamacrocyclophosphonic acid complex as claimed in Claim 14 or Claim 15, with the proviso that one of R+, R7 or R® must be present, and covalently attached to a dextran, a polypeptide or a molecule that has specific affinity for a receptor.

20. A conjugate as claimed m claim 19, wherein the molecule with a specific affinity for a receptor is an antibody or antibody fragment.

21. A conjugate of Claim 20 wherein the antibody or antibody fragment is a monoclonal antibody or fragmeint thereof.

22. A conjugate of any one of Claims 19 to 21 wherein A is CH, and one of Q or Z is CH and the other is C-C(0)-R6, where R6 is NHR7, where R7 is a biologically active material.

23. A pharmaceutical formulation comprising a complex of any one of Claims 14 to 18 with a pharmaceutically acceptable carrier.

24. A pharmaceutical formulation comprising a conjugate of any one of Claims 19 to 22 with a pharmaceutically acceptable carrier.

25. The complex as claimed in any one of Claims 14 tc 18 for use as a pharmaceutical.

26. The conjugate as claimed in any one of Claims 19 to 22 for use as a pharmaceutical.

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25 2 5 5 4

27. A kit for use as a diagnostic agent having as an ingredient a ligand as claimed in any one of Claims 1 to 13.

28. A process for preparing a complex as claimed in Claim 14 which comprises reacting a bicyclopolyazamacrocyclophosphonic acid compound as claimed in Claim 1 with a metal ion selected from Gd+3, Mn42 or Fe"^ under aqueous conditions at a pH from 5 to 7.

29. A process for preparing a bicyclopolyazamacrocyclophosphonic acid compound as claimed in Claim 1 which comprises reacting:

(A) a compound of the Formula (I) wherein at least 1 R group is H, with a phosphonating agent; or

(B) a compound of Formula (I) wherein Q, A or Z has a protecting group present, after step (A), removing the blocking group by catalytic hydiogenation or acid hydrolysis.

30. A process of Claim 29 wherein the phosphonating agent has the formula P(0R)j where R is defined as in Claim 1.

31. A process of Claim 29 wherein the phosphonating agent has the formula P(OR)3 where R is defined as in Claim 1, and formaldehyde is a solvent.

32. 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.

33. 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 24.

34. A compound of the formula (I) as defined in claim 1 substantially as herein described with reference to any example thereof.

35. A complex as defined in claim 14 substantially as herein described with reference to any example thereof.

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^^36. A conjugate as defined in claim 19 substantially as herein described with reference to any example thereof.

37. A pharmaceutical formulation as defined in claim 23 or 24 substantially as herein described with reference to any example thereof.

38. A process as defined in claim 28 or claim 29 substantially as herein described with reference to any example thereof.

39. A method as defined in claim 32 or 33 substantially as herein described with reference to any example thereof.

end of claims

N.Z FW6NfoFRc£~)

2 7 NOV 1997

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