Bicyclopol yazamacrocyclophosphonic acid complexes for use as contrast agents

Bicyclopolyazamacrocyclophosphonic acid compounds are disclosed which may form inert complexes with Gd, Mn or Fe ions. The overall charge of the complex can be varied to alter the in vivo biolocalization. Such complexes can be covalently attached to an antibody, antibody fragment or other biologically active molecule to form conjugates. The complexes and conjugates are useful as contrast agents for diagnostic purposes.

FIELD OF THE INVENTION 
This invention concerns ligands that are bicyclopolyazamacrocyclophosphonic 
acids, and complexes and conjugates thereof, for use as contrast agents in 
magnetic resonance imaging (MRI). Some ligands and complexes are also 
useful as oral care agents and as scale inhibiting agents in water 
treatment systems. To better understand this invention, a brief background 
on MRI is provided in the following section. 
BACKGROUND OF THE INVENTION 
MRI is a non-invasive diagnostic technique which produces well resolved 
cross-sectional images of soft tissue within an animal body, preferably a 
human body. This technique is based upon the property of certain atomic 
nuclei (e.g. water protons) which possess a magnetic moment as defined by 
mathematical equations; see G. M. Barrow, Physical Chemistry, 3rd Ed., 
McGraw-Hill, N.Y. (1973)! to align in an applied magnetic 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 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 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 electrons producing 
the magnetic moment Magnetic Resonance Annual, 23-266, Raven Press, N.Y. 
(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 
paramagnetic chelates be administered to animals in order to increase the 
diagnostic information obtained by MRI Frontiers of Biol. Energetics I, 
752-759 (1978); J. Nucl. Med. 25, 506-513 (1984); Proc. of NMR Imaging 
Symp. (Oct. 26-27, 1980); F. A. Cotton et al., 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.sup.+3), iron 
(Fe.sup.+3), manganese (Mn.sup.+2) and (Mn.sup.+3), and chromium 
(Cr.sup.+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. 
GdCl.sub.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. Pat. No. 
4,899,755 discloses a method of alternating the proton NMR relaxation 
times in the liver or bile duct of an animal using Fe.sup.+3 
-ethylene-bis(2-hydroxyphenylglycine) complexes and its derivatives, and 
suggests among various other compounds the possible use of a pyridine 
macrocyclomethylenecarboxylic acid. U.S. Pat. No. 4,880,008 (a CIP of U.S. 
Pat. No. 4,899,755) discloses additional imaging data for liver tissue of 
rats, but without any additional complexes being shown. U.S. Pat. No. 
4,980,148 disclose gadolinium complexes for MRI which are non-cyclic 
compounds. C. J. Broan et al., J. Chem. Soc., Chem. Commun., 1739-1741 
(1990) describe some bifunctional macrocyclic phosphinic acid compounds. 
C. J. Broan et al., J. Chem. Soc., Chem. Commun., 1738-1739 (1990) 
describe compounds that are triazabicyclo compounds. I. K. Adzamli et al., 
J. Med. Chem. 32, 139-144 (1989) describes acyclic phosphonate derivatives 
of gadolinium complexes for NMR imaging. 
At the present time, the only commercial contrast agents available in the 
U.S. are the complex of gadolinium with diethylenetriaminepentaacetic acid 
(DTPA-Gd.sup.+3 -MAGNEVIST.TM. by Shering) and a DO3A derivative 
1,4,7-tris(carboxymethyl)-10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclodode 
canato!-gadolinium (PROHANCE.TM. by Squibb). MAGNEVIST.TM. and PROHANCE.TM. 
are each considered as a non-specific/perfusion agent since it freely 
distributes in extracellular fluid followed by efficient elimination 
through the renal system. MAGNEVIST.TM. 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.TM., Guerbet is 
commercially marketing a macrocyclic perfusion agent (DOTAREM.TM.) which 
presently is only available in Europe. PROHANCE.TM. is shown to have fewer 
side effects than Magnevist.TM.. A number of other potential contrast 
agents are in various stages of development. 
SUMMARY OF THE INVENTION 
Surprisingly, it has now been found that various 
bicyclopolyazamacrocyclophosphonic acid ligands can be contrast agents. 
Furthermore, these ligands may have their 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 bicyclopolyazamacrocyclophosphonic acid 
compounds of the formula 
##STR1## 
wherein: 
##STR2## 
where: X and Y are independently H, OH, C.sub.1 -C.sub.3 alkyl or COOH; 
n is an integer of 1, 2 or 3; 
with the proviso that: when n is 2, then the sum of X and Y must equal two 
or more H; and when n is 3, then the sum of X and Y must equal three or 
more H; 
T is H, C.sub.1 -C.sub.18 alkyl, COOH, OH, SO.sub.3 H, 
##STR3## 
where: R.sup.1 is OH, C.sub.1 -C.sub.5 alkyl or --O--(C.sub.1 -C.sub.5 
alkyl); 
R.sup.4 is H, NO.sub.2, NH.sub.2, isothiocyanato, semicarbazido, 
thiosemicarbazido, maleimido, bromoacetamido or carboxyl; 
R.sup.2 is H or OH; with the proviso that when R.sup.2 is OH, then the R 
term containing the R.sup.2 must have all X and Y equal to H; 
with the proviso that at least one T must be P(O)R.sup.1 OH, and with the 
proviso that when one T is 
##STR4## 
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.sup.3, C--OR.sup.8, N.sup.+ -R.sup.5 
X.sup.-, 
##STR5## 
R.sup.3 is H, C.sub.1 -C.sub.5 alkyl, benzyl, or benzyl substituted with 
at least one R.sup.4 ; 
R.sup.4 is defined as above; 
R.sup.5 is C.sub.1 -C.sub.16 alkyl, benzyl, or benzyl substituted with at 
least one R.sup.4 ; 
R.sup.8 is C.sub.1 -C.sub.16 alkylamino; 
X.sup.- is Cl.sup.-, Br.sup.-, I.sup.- or H.sub.3 CCO.sub.2.sup.- ; 
Q and Z independently are CH, N, N.sup.+ -R.sup.5 X.sup.-, C--CH.sub.2 
--OR.sup.3 or C--C(O)--R.sup.6 ; 
R.sup.5 is defined as above; 
R.sup.6 is --O--(C.sub.1 -C.sub.3 alkyl), OH or NHR.sup.7 ; 
R.sup.7 is C.sub.1 -C.sub.5 alkyl or a biologically active material; 
X.sup.- is defined as above; or 
pharmaceutically-acceptable salts thereof; 
with the proviso that: 
a) when Q, A or Z is N or N.sup.+ --R.sup.5 X.sup.-, then the other two 
groups must be CH; 
b) when A is C--Br, C--Cl, C--OR.sup.3 or C--OR.sup.8 then both Q and Z 
must be CH; 
c) the sum of the R.sup.4, R.sup.7 and R.sup.8 terms, when present, may not 
exceed one; and 
d) only one of Q or Z can be C--C(O)--R.sup.6 and when one of Q or Z is 
C--C(O)--R.sup.6, then A must be CH. 
When the above ligands of Formula (I) have at least two of the R terms T 
equal to PO.sub.3 H.sub.2 P(O)R.sup.1 OH where R.sup.1 is OH! and the 
third T equal H, COOH or C.sub.1 -C.sub.18 alkyl; A, Q and Z are CH; n is 
1; and X and Y independently are H or C.sub.1 -C.sub.3 alkyl; then the 
ligands are useful for oral care. Particularly preferred are those ligands 
where in the three R terms T is P(O)R.sup.1 OH, where R.sup.1 is OH; n is 
1; and X and Y are H. The use of these ligands is discussed in our 
copending U.S. patent applications Ser. No. 805,600, filed Dec. 10, 1991, 
entitled "Oral Compositions for Inhibiting Calculus" by R. K. Frank, J. R. 
Garlich, J. Simon, G. E. Kiefer and D. A. Wilson (Attorney Docket No. 
C-38, 337) and Ser. No. 805,598, filed Dec. 10, 1991, and entitled "Oral 
Compositions for Inhibiting Plaque Formation" by J. R. Garlich, R. K. 
Frank, J. Simon, G. E. Kiefer and D. A. Wilson (Attorney Docket No. C-39, 
198), the disclosures of which are hereby incorporated by reference. 
When the above ligands of Formula (I) have: 
in the R term at least two T equal P(O)R.sup.1 OH, where R.sup.1 is OH, and 
in the other R term, T is COOH or P(O)R.sup.1 OH and n, R.sup.1, X, Y, A, 
Q and Z are defined as above; 
in at least one R term T is P(O)R.sup.1 OH, where R.sup.1 is OH, and in the 
other two R terms, T is COOH or P(O)R.sup.1 OH, and n, R.sup.1, X, Y, A, Q 
and Z are defined as above; or 
in the R term three T equal P(O)R.sup.1 OH, where R.sup.1 is C.sub.1 
-C.sub.5 alkyl or --O--(C.sub.1 -C.sub.5 alkyl) and n, R.sup.1, X, Y, A, Q 
and Z are defined as above; 
then the ligands are useful as contrast agents. 
Particularly preferred are those ligands of Formula (I) where: 
X and Y are H; 
n is 1; or 
A, Q and Z are CH. 
Preferably the ligands and complexes of Formula (I) do not have all three T 
equal to PO.sub.3 H.sub.2 P(O)R.sup.1 OH where R.sup.1 is OH! when A, Q 
and Z are CH; although such complexes are useful as contrast agents or 
oral care agents. 
Bifunctional ligands of Formula (I) are desirable to prepare the conjugates 
of this invention. Such ligands must have: 
one R term where the T moiety is 
##STR6## 
where R.sup.2 and R.sup.4 are defined as above, especially where in the 
two R terms not containing an R.sup.4 term, both T terms are P(O)R.sup.1 
OH, where R.sup.1 is defined as above or where in the two R terms not 
containing an R.sup.4 term, one T term is a COOH and the other T term is 
P(O)R.sup.1 OH, where R.sup.1 is defined as above; preferably that moiety 
of the above T term where one of X or Y of that term is COOH; and also 
preferred are those ligands where n is 1 and/or the remaining X and Y 
terms are H; or 
A is C--OR.sup.3 or C--OR.sup.8 where R.sup.3 and R.sup.8 are defined as 
above or 
##STR7## 
where R.sup.4 is defined as above; or A is CH, and one of Q or Z is CH and 
the other is C--C(O)--R.sup.6 where R.sup.6 is defined as above; 
especially those ligands where R.sup.6 is NHR.sup.7, where R.sup.7 is a 
biologically active material. 
The ligands of Formula (I) may be complexed with various metal ions, such 
as gadolinium (Gd.sup.+3), iron (Fe.sup.+3), and manganese (MN.sup.+2), 
with Gd.sup.+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 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: 
(A) an overall charge of -2 or more--when in three R terms T is P(O)R.sup.1 
OH, where R.sup.1 is OH, and n is 1; 
in two R terms T is P(O)R.sup.1 OH, where R.sup.1 is OH, in the third R 
term T is COOH, and n is 1; 
in two R terms T is P(O)R.sup.1 OH, where R.sup.1 is OH, in the third R 
term T is P(O)R.sup.1 OH, where R.sup.1 is C.sub.1 -C.sub.5 alkyl, and n 
is 1; or 
in two R terms T is P(O)R.sup.1 OH, where R.sup.1 is OH, in the third R 
term T is P(O)R.sup.1 OH, where R.sup.1 is --O--(C.sub.1 -C.sub.5 alkyl), 
and n is 1; 
(B) an overall charge of -1--when 
in one R term T is P(O)R.sup.1 OH, where R.sup.1 is OH, and in the other 
two R terms T is P(O)R.sup.1 OH, where R.sup.1 is --O--(C.sub.1 -C.sub.5 
alkyl), and n is 1; 
in one R term T is P(O)R.sup.1 OH, where R.sup.1 is OH, and in the other 
two R terms T is P(O)R.sup.1 OH, where R.sup.1 is C.sub.1 -C.sub.5 alkyl, 
and n is 1; or 
in one R term T is P(O)R.sup.1 OH, where R.sup.1 is OH, and in the other 
two R terms T is COOH, and n is 1; 
(C) an overall neutral charge-when 
in the three R terms T is P(O)R.sup.1 OH, where R.sup.1 is --O--(C.sub.1 
-C.sub.5 alkyl), and n is 1; or 
in the three R terms T is P(O)R.sup.1 OH, where R.sup.1 is C.sub.1 -C.sub.5 
alkyl, and n is 1; or 
(D) an overall charge of +1--when 
one of A, Q or Z is N.sup.+ --R.sup.5 X.sup.-, where R.sup.5 and X.sup.- 
are defined as above; and in one R term, the T moiety is P(O)R.sup.1 OH, 
where R.sup.1 is C.sub.1 -C.sub.5 alkyl or --O--(C.sub.1 -C.sub.5 alkyl); 
and in the other two R terms, the T moiety is COOH or P(O)R.sup.1 OH, 
where R.sup.1 is C.sub.1 -C.sub.5 alkyl, --O--(C.sub.1 -C.sub.5 alkyl); 
and all X and Y terms are H. 
Both the complexes and conjugates may be formulated to be in a 
pharmaceutically acceptable form for administration 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 copending U.S. patent application Ser. No. 
806,069, filed Dec. 10, 1991, entitled "Bicyclopolyazamacrocyclophosphonic 
Acid Complexes, and Conjugates Thereof, for Use as Radiopharmaceuticals" 
by G. E. Kiefer and J. Simon (Attorney Docket No. C-39, 771), filed on 
even date herewith, the disclosure of which is hereby incorporated by 
reference. 
DETAILED DESCRIPTION OF THE INVENTION 
The compounds of Formula (I) are numbered for nomenclature purposes as 
follows: 
##STR8## 
One aspect of the present invention concerns development of contrast agents 
having synthetic modifications to the paramagnetic chelate enabling site 
specific delivery of the contrast agent to a desired tissue. The advantage 
being increased contrast in the areas of interest based upon tissue 
affinity as opposed to contrast arising from non-specific perfusion which 
may or may not be apparent with an extracellular agent. The specificity of 
the ligand of Formula (I) may be controlled by adjusting the total charge 
and lipophilic character of the complex. The overall range of the charge 
of the complex is from -3 to +1. For example, for a complex having 2 or 
more PO.sub.3 H.sub.2 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. Pat. Nos. 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 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.sup.+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. possibly -2 or -3 for bone, -1 for 
liver, or +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.sub.1 -C.sub.3 alkyl", "C.sub.1 -C.sub.5 alkyl", "C.sub.1 
-C.sub.18 alkyl", include both straight and branched chain alkyl groups. 
An "animal" includes a warmblooded mammal, preferably a human being. 
"Biologically active material" refers to a dextran, peptide, or molecules 
that have specific affinity for a receptor, or preferably antibodies or 
antibody fragments. 
"Antibody" refers to any polyclonal, monoclonal, chimeric antibody or 
heteroantibody, preferably a monoclonal antibody; "antibody fragment" 
includes Fab fragments and F(ab').sub.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 1116-NS-19-9 (anti-colorectal 
carcinoma), 1116-NS-3d (anti-CEA), 703D4 (anti-human lung cancer), 704A1 
(anti-human lung cancer), CC49 (anti-TAG-72), CC83 (anti-TAG-72) and 
B72.3. The 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 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 a antibody or antibody 
fragment. The terms "bifunctional coordinator", "bifunctional chelating 
agent" and "functionalized chelant" are used interchangeably and refer to 
compounds that have a chelant moiety capable of chelating a metal ion and 
a moiety covalently bonded to the chelant moiety that is capable of 
serving as a means to covalently attach to an antibody or antibody 
fragment. 
The bifunctional chelating agents described herein (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 
R.sup.4 or R.sup.8 in Formula I), can be covalently attached to 
biologically active materials, such as dextran, molecules that have 
specific affinity for a receptor, or preferably covalently attached to 
antibodies or antibody fragments. 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 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, 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 ammonium or 1-deoxy-1-(methylamino)-D-glucitol, alkali metal ions, 
alkaline earth metal ions, and other similar ions. Particularly preferred 
are the salts of the compounds of Formula (I) where the salt is potassium, 
sodium, ammonium. Also included are mixtures of the above salts. 
DETAILED DESCRIPTION OF THE PROCESS 
The compounds of Formula (I) are prepared by various processes. Further 
discussion of suitable processes to make the compounds of Formula (I) are 
in our copending U.S. patent application Ser. No. 08/058,101, filed May 6, 
1993, entitled "Process for the Preparation of Azamacrocyclic or Acyclic 
Aminophosphonate Ester Derivatives" by G. E. Kiefer (Attorney Docket No. 
C-41, 184), filed on even date herewith, the disclosure of which is hereby 
incorporated by reference. 
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.dbd.H, n=1 (but would also apply if n=2 or 3 with the corresponding 
change in the reagent), T.dbd.PO.sub.3 H.sub.2, and Q, A and Z.dbd.CH. 
##STR9## 
Scheme 2 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR10## 
where R.sup.1 =--O--(C.sub.1 -C.sub.5 alkyl); and Q, A and Z.dbd.CH. 
##STR11## 
Scheme 3 Prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR12## 
where R.sup.1 =C.sub.1 -C.sub.5 alkyl; and Q, A and Z.dbd.CH. 
##STR13## 
Scheme 4 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR14## 
where R.sup.1 =--O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 alkyl; 
A.dbd.C--Br, and Q and Z.dbd.CM. 
##STR15## 
Scheme 5 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR16## 
where R.sup.1 =--O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 alkyl; 
##STR17## 
R.sup.4 .dbd.H, NO.sub.2, NH.sub.2 or SCN; and Q and Z.dbd.CH. 
##STR18## 
Scheme 6 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR19## 
where R.sup.1 =--O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 alkyl; 
A=C--OR.sup.8, where R.sup.8 .dbd.C.sub.1 -C.sub.5 alkylamino; and Q and 
Z.dbd.CH. 
##STR20## 
Scheme 7 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR21## 
where R.sup.1 =--OH, --O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 
alkyl; 
Z.dbd.C--C(O)--R.sup.6 where R.sup.6 .dbd.OH; and Q and A.dbd.CH. 
##STR22## 
Scheme 8 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR23## 
where R.sup.1 =--OH, --O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 
alkyl; 
Z.dbd.C--CH.sub.2 --OR.sup.3 where R.sup.3 =benzyl; and 
Q and A.dbd.CH. 
##STR24## 
Scheme 9 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR25## 
where R.sup.1 =--OH, --O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 
alkyl; 
A.dbd.N or N--R.sup.5 ; R.sup.5 .dbd.C.sub.1 -C.sub.16 alkyl halide; and 
Q and Z.dbd.CH. 
##STR26## 
Scheme 10 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR27## 
where R.sup.1 =--OH, --O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 
alkyl; 
Q.dbd.N--R.sup.5 ; R.sup.5 =C.sub.1 -C.sub.16 alkyl halide; and 
A and Z.dbd.CH. 
##STR28## 
Scheme 11 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), 
##STR29## 
where R.sup.1 =--OH, --O--(C.sub.1 -C.sub.5 alkyl) or C.sub.1 -C.sub.5 
alkyl; 
Q.dbd.N or N--R.sup.5 R.sup.5 .dbd.C.sub.1 -C.sub.16 alkyl halide; and 
A and Z.dbd.CH. 
##STR30## 
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 formaldehyde sodium bisulfite addition to give 
quantitative conversion to the 4,9-substituted sulfonate derivative, which 
is then converted to the corresponding nitrile. Subsequent 
phosphonomethylation and hydrolysis yields the desired product. 
Scheme 12 prepares the compounds of Formula (I) wherein X and Y.dbd.H, n=1 
(but would also apply if n=2 or 3 with the corresponding change in the 
reagent), R at the 3 position has 
##STR31## 
where R.sup.1 =--OH or --O--(C.sub.1 -C.sub.5 alkyl); and the other two R 
terms have T.dbd.COOH; and 
A, Q and Z.dbd.CH. 
##STR32## 
Scheme 13 prepares the compounds of Formula (I) wherein X and Y.dbd.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 
##STR33## 
where R.sup.1 =OH or --O--(C.sub.1 -C.sub.5 alkyl); and the other R term 
at the 9 position has T.dbd.COOH; and 
A, Q and Z.dbd.CH. 
##STR34## 
Scheme 14 prepares the compounds of Formula (I) wherein X and Y.dbd.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 have 
##STR35## 
Scheme 15 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 9 positions have 
##STR36## 
where R.sup.1 =--OH or --O--(C.sub.1 -C.sub.5 alkyl); and X and Y.dbd.H; 
the R term at the 6 position has 
##STR37## 
where R.sup.4 .dbd.NO.sub.2 or NH.sub.2 ; and one of X or Y.dbd.H and the 
other=COOH; and 
A, Q and Z.dbd.CH. 
##STR38## 
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 
##STR39## 
where R.sup.1 =--OH or --O--(C.sub.1 -C.sub.5 alkyl); and X and Y.dbd.H; 
the R term at the 9 position has 
##STR40## 
where R.sup.4 .dbd.NO.sub.2 or NH.sub.2 ; and one of X or Y.dbd.H and the 
other=COOH, 
A, Q and Z.dbd.CH. 
##STR41## 
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 
##STR42## 
where R.sup.1 =--OH; and X and Y.dbd.H; 
the R term at the 3 and 9 positions have T.dbd.COOH; and 
A, Q and Z.dbd.CH. 
##STR43## 
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 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 
similarly reactive product which would work well in subsequent ring 
closure reactions. Macrocyclization procedures are numerous in the 
literature and the desired tetraazamacrocycle (3) was prepared according 
to the method of Stetter et al., Tetrahedron 37, 767-772 (1981). More 
general procedures have since been published which give good yields of 
similar macrocycles using milder conditions A. D. Sherry et al., J. Org. 
Chem. 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. 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 phosphonic acid 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 nucleophilic 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.degree. 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 presence of a solvent. When alcohols are employed as the 
solvent for either dialkyl or trialkyl 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 .alpha.-halo-dialkylphosphonates in solvents such as 
acetonitrile, chloroform, dimethylformamide, tetrahydrofuran or 
1,4-dioxane with or without the addition of a non-nucleophilic base such 
as potassium carbonate at room temperature or above. The resulting 
perester intermediate is then readily hydrolyzed under basic conditions 
(aqueous hydroxide, pH=8-14, 30.degree.-110.degree. C.) to give the 
corresponding half-acid derivative. 
In Scheme 3, macrocyclic methylphosphinic acids (10 and 11) are prepared 
under conditions similar to those described in Scheme 2. Using 
diethoxymethylphosphine as the nucleophilic species and paraformaldehyde, 
condensation can be conducted in solvents such as tetrahydrofuran, 
dimethylformamide, dioxane, acetonitrile or alcholic media. The resulting 
phosphinate ester is then hydrolyzed under acid (6N HCl, 
80.degree.-100.degree. C.) or basic (stoichiometric quantities of base, 
40.degree.-100.degree. C.) conditions to give the corresponding 
methylphosphonic acid. Alternatively, the method devised by A. D. Sherry 
et al. (Inorg. Chem., submitted 1991) using ethylphosphonic acid generated 
in situ can be used to obtain phosphinate derivatives having increased 
lipophilic character. 
Scheme 4 illustrates an approach to incorporate additional functionality 
into the pyridine unit of the 12-membered tetraazamacrocycle. Thus, 
chelidamic acid (Sigma Chemical Company; 12) can be converted to the 
bis-halomethyl derivative (13) having appropriate substitution at the 
pyridyl 4-position. Transformations leading to this intermediate are 
general in nature and its preparation is described by Takalo et al. Acta 
Chemica Scandinavica B 42, 373-377 (1988)!. Subsequent macrocyclization 
using this intermediate (15) can be accomplished by the standard DMF 
reaction at 100.degree. C. with the sodiotritosylated triamine, or at room 
temperature with the tritosylated free base and potassium carbonate, 
sodium carbonate, or cesium carbonate as base to give products similar to 
those previously described. Subsequent reactions leading to phosphonate 
half-acids and phosphinate functionality are identical to those 
transformations and conditions described in the preceeding Schemes. 
In Scheme 4, 4-halopyridyl substituted macrocycles (16) are described which 
can undergo substitution at the 4-position of the pyridyl moiety as 
described in Scheme 5. Thus, organometallic Pd(II) complexes can be 
employed to facilitate the coupling reaction between phenylacetylene and 
phenylacetylene derivatives and the pyridyl macrocycle. Typical reaction 
conditions for this transformation utilize anhydrous conditions with 
triethylamine as solvent and at reaction temperature between about 
10.degree. to about 30.degree. C. for optimum yields. The identical 
product can also be obtained using Cu(I) phenylacetylide in anhydrous 
pyridine at a temperature between about 80.degree. to about 110.degree. C. 
In addition, standard anionic alkylation procedures can be employed to 
affect substitution on the pyridine nucleus with, for example, 
sodioalkoxides in DMF or dioxane at from about 80.degree. to about 
100.degree. C. using bases such as potassium carbonate or sodium 
hydroxide. Macrocyclic tetraazamacrocycles (24, 25, 26, 27, 28) dervatized 
in this manner are compatible with transformations described in previous 
Schemes resulting in analogous phosphonate chelants. 
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 macrocyclic structure. This type of alkylation 
procedure is best accomplished under anhydrous conditions in an aprotic 
solvent such as tetrahydrofuran (THF) and using a non-nucleophilic base 
such as sodium hydride or butyllithium at temperatures between from about 
-30.degree. to about 80.degree. C. The generality of this approach has 
been described by Chaubet et al., for acyclic analogs Tetrahedron Letters 
31 (40), 5729-5732 (1990)!. The macrocyclic nitrile prepared in this 
manner can be reduced to the primary amine (36) by standard procedures 
followed by protection of the primary amine with 
2-(t-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON; 37). Subsequent 
functionalization of the macrocyclic 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. 
Functionalization can also be carried out on the 3-position of the pyridine 
ring within the macrocyclic structure as illustrated in Scheme 7. Newkome 
et al. Tetrahedron 39(12), 2001-2008 (1983)! has previously described the 
synthesis of ethyl 2,6-halomethylnicotinate (45) which serves as the 
initial starting material in this synthetic route. Thus, the 
tris-tosylated macrocycle intermediate (46) can be detosylated under 
acidic conditions (HBr/AcOH, 25.degree.-115.degree. C.) with simultaneous 
hydrolysis to yield the nicotinic acid derivative (48), or reduction of 
the ester in refluxing ethanol prior to detosylation will result in the 
3-hydroxymethyl intermediate (47). The nicotinic acid macrocycle can then 
be substituted into the general scheme for secondary amine 
functionalization to yield the various types of phosphonate chelants of 
Formula (I) (49, 50, 51, 52, 53). 
In contrast, the 3-hydroxymethyl analog is advantageously protected prior 
to functionalization of the macrocyclic amines. The benzyl (Bz) protecting 
group is shown in Scheme 8 since it must be resistant to the severe acid 
conditions encountered in the detosylation step. After appropriate 
functionalization of the secondary amines has been accomplished as 
described in previous Schemes, the benzyl group is removed under mild 
catalytic hydrogenation conditions (58). 
Macrocyclic derivatives can also be prepared as in Schemes 12-14 where both 
carboxylate and phosphonate chelating fuctionalities are present in the 
same molecule. Thus, varying degrees of carboxylate fuctionality can be 
introduced under typical aqueous alkylation procedures using bromoacetic 
acid. Following this step, the remaining amines can be phosphonomethylated 
by procedures discussed in previous Schemes using formaldehyde and 
phosphorous acid, dialkyl phosphonates or trialkyl phosphites. 
Schemes 15 and 16 delineate a synthetic approach which introduces an 
aromatic nitrobenzyl substituent at one of the macrocyclic nitrogen 
positions. Typically, the macrocyclic amine is mono-N-functionalized in an 
organic solvent such as acetonitrile or DMF at room temperature using a 
non-nucleophilic base such as potassium carbonate. Additional 
functionalization of the remaining nitrogen positions is then performed by 
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. 
Scheme 17 illustrates the synthesis of the macrocyclic compounds (4) where 
the amines at positions 3 and 9 are reacted with at least two moles of the 
sodium salt of hydroxymethanesulfonic acid in water at a pH of about 9 to 
provide the corresponding macrocyclic compound where positions 3 and 9 are 
the sodium salt of methanesulfonic acid (119). The sulfonic acid group is 
then displaced using sodium cyanide to form the corresponding cyanomethane 
derivative (120). The cyano group is hydrolyzed to the carboxylic acid 
either: simultaneously with the addition of phosphorous acid and 
formaldehyde; or by sequential reaction with a derivative of phosphorous 
acid and formaldehyde to form the phosphonic acid at the 6 position (121), 
followed by acid hydrolysis, at an elevated temperature, of the cyanato 
groups and any derivative moiety of the phosphorous acid present. The 
resulting compound is a macrocycle with two carboxylic acid groups at 
positions 3 and 9 and a phosphonic acid group at position 6. The 
phosphonomethylation can also be preformed by the methods discussed above. 
The metal ions used to form the complexes of this invention are Gd.sup.+3, 
Mn.sup.+2, Fe.sup.+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 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.sup.+3), iron (Fe.sup.+3) and manganese (MN.sup.+2), with 
Gd.sup.+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 bicyclopolyazamacrocyclophosphonic 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, Kohler and Milstein 
Nature, 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 in 
the conjugates are monoclonal antibodies, or fragments thereof having high 
specificity for a desired epitope(s). Antibodies used in the present 
invention may be directed against, for example, tumors, bacteria, fungi, 
viruses, parasites, mycoplasma, differentiation and other cell membrane 
antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and 
any biologically active molecules. Some examples of antibodies or antibody 
fragments are 1116-NS-19-9, 1116-NS-3d, 703D4, 704A1, CC49, CC83 and 
B72.3. All of these antibodies have been deposited in ATCC. A more 
complete list of antigens can be found in U.S. Pat. No. 4,193,983, which 
is incorporated herein by reference. The conjugates of the present 
invention are particularly preferred for the diagnosis of various cancers. 
This invention is used with a physiologically acceptable carrier, excipient 
or vehicle therefore. The methods for preparing such formulations are well 
known. The formulations may be in the form of a suspension, injectable 
solution or other suitable formulations. Physiologically acceptable 
suspending media, with or without adjuvants, may be used. 
An "effective amount" of the formulation is used for diagnosis. The dose 
will vary depending on the disease and physical parameters of the animal, 
such as weight. In vivo diagnostics are also contemplated using 
formulations of this invention. 
Other uses of some of the chelants of the present invention may include the 
removal of undesirable metals (i.e. iron) from the body, attachment to 
polymeric supports for various purposes, e.g. as diagnostic agents, and 
removal of metal ions by selective extraction. The ligands of Formula (I) 
having in at least two R terms T equal to P(O)R.sup.1 OH may be used for 
metal ion control as scale inhibitors. Some of these ligands can be used 
in less than stoichiometric amounts. Similar uses are known for compounds 
described in U.S. Pat. Nos. 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. 
Some terms used in the following examples are defined as follows: 
LC=liquid chromatrography, purifications were carried out at low pressure 
using Dionex 2010i system fitted with a hand-packed Q-Sepharose.TM. anion 
exchange column (23.times.2 cm). 
DMF=dimethylforamide. 
AcOH=acetic acid. 
ICP=inductively coupled plasma. 
g=gram(s). 
mg=milligrams. 
kg=kilogram(s). 
mL=milliliter(s). 
.mu.L=microliter(s). 
pH Stability General Procedure 
A stock .sup.159 GdCl.sub.3 (or .sup.153 SmCl.sub.3) solution was prepared 
by adding 2 .mu.L of 3.times.10.sup.-4 M .sup.159 GdCl.sub.3 in 0.1N HCl 
to 2 mL of a 3.times.10.sup.-4 M GdCl.sub.3 carrier solution. Appropriate 
ligand solutions were then prepared in deionized water. The 1:1 
ligand/metal complexes were then prepared by combining the ligands 
(dissolved in 100-500 .mu.L of deionized water) with 2 mL of the stock 
.sup.159 GdCl.sub.3 solution, followed by through mixing to give an acidic 
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.TM. G-50 column, eluting 
with 4:1 saline (85% NaCl/NH.sub.4 OH) and collecting 2.times.3 mL 
fractions. The amount of radioactivity in the combined elutions was then 
compared with that left on the resin (non-complexed metal is retained on 
the resin). The pH stability profile was generated by adjusting the pH of 
an aliquot of the complex solution using 1M NaOH or 1M HCl and determining 
the percent of the metal existing as a complex using the ion exchange 
method described above. The Sm results are known by experimental 
comparison to be identical for complexation and biodistribution of the 
ligands of this invention. 
STARTING MATERIALS

EXAMPLE A 
Preparation of 2,6-bis(chloromethyl)pyridine 
To 100 mL of thionyl chloride that was cooled (ice bath) was added 24 g 
(0.17 mol) of 2,6-bis(hydroxy-methyl)pyridine. After 30 min, the reaction 
mixture was warmed to room temperature, then refluxed for 1.5 hrs. 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 NaHCO.sub.3, filtered and dried to yield 23.1 g 
(71.5%) of the titled product as an off-white crystalline solid, mp 
74.5.degree.-75.5.degree. C., and further characterized by: 
.sup.1 H NMR (CDCl.sub.3) .delta.4.88 (s, 4H), 7.25-7.95 (m, 3H). 
EXAMPLE B 
Preparation of 
3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15) 
,11,13-triene 
A DMF solution (92 mL) of 6.9 g (11.4 mmol) of 
1,4,7-tris(p-tolylsulfonyl)diethylenetriamine disodium salt was stirred 
and heated to 100.degree. C. under nitrogen. To the solution was added 
dropwise over 45 min 2 g (11.4 mmol) of 2,6-bis(chloromethyl)pyridine 
(prepared by the procedure of Example A) in 37 mL of DMF. When the 
addition was completed the reaction mixture was stirred at 40.degree. C. 
for 12 hrs. To the reaction mixture was then added 50-75 mL of water, 
resulting in immediate dissolution of NaCl, followed by precipitation of 
the title product. The resulting 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.degree.-170.degree. C. dec. and 
further characterized by: 
.sup.1 H NMR (CDCl.sub.3) .delta.2.40 (s, 3H), 2.44 (s, 6H), 2.75 (m, 4M), 
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 
.sup.13 C NMR .delta.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-tetraazabicyclo9.3.1!pentadeca-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-tetraazabicyclo9.3.1!pentadeca-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 vigorously and 15-20 mL of 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 light-tan oil which gradually 
crystallized upon standing at room temperature to yield 1.2 g (71%) of the 
title product, mp 86.degree.-88.degree. C. and further characterized by: 
.sup.1 H NMR (CDCl.sub.3) .delta.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 
.sup.13 C NMR .delta.48.73, 49.01, 53.63, 119.67, 136.29, 159.54. 
EXAMPLE D 
Preparation of 3,9-bis(sodium 
methylenesulfonate)-3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-t 
riene (PC2S) 
An aqueous solution (10.0 mL) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-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. To the solution was then added 
1.37 g (10.2 mmol) of HOCH.sub.2 SO.sub.3 Na with 5 mL of deionized water. 
The solution was heated at 60.degree. C. for 10 min and the pH dropped 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: 
.sup.1 H NMR (D.sub.2 O) .delta.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 
.sup.13 C NMR (D.sub.2 O) .delta.48.52, 54.04, 58.92, 79.09, 123.90, 
141.37, 161.89. 
EXAMPLE E 
Preparation of 
3,9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),1 
1,13-triene 
To an aqueous solution, 10.0 mL, of 3,6,9-bis(sodium 
methylenesulfonate)-3,6,9,15-tetraaza-bicyclo9.3.1!pentadeca-1(15),11,13- 
triene (prepared by the procedure of Example D), 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 extracted with chloroform (3.times.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: 
.sup.1 H NMR (CDCl.sub.3) .delta.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 
.sup.13 C NMR (CDCl.sub.3) .delta.46.08, 46.64, 52.89, 60.78, 
115.31,122.02, 137.57, 157.33. 
EXAMPLE F 
Preparation of 
3,9-bis(methylenenitrile)-6-(methylenedimethylphosphonate)-3,6,9,15-tetraa 
zabicyclo9.3.1!pentadeca-1(15),11,13-triene 
3,9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11 
,13-triene (prepared by the procedure of Example E), 285 mg (1.0 mmol) was 
combined with 60 mg (2.0 mmol, excess) of paraformaldehyde and 0.354 mL 
(372 mg, 3.0 mmol, excess) of trimethylphosphite. The mixture was gently 
stirred for 10 min to obtain a slurry, then heated to 90.degree. C. for 1 
hr. After the excess reagents and byproducts were removed in vacuo (1 hr 
at 125.degree. C./0.01 mmHg), he resulting dark brown residue was 
dissolved in 20 mL of chloroform and washed with deionized water 
(5.times.15 mL). The organic layer was dried over anhydrous magnesium 
sulfate, filtered, and the excess solvents evaporated in vacuo to give the 
desired product as a yellow waxy solid, 168 mg (417) and characterized by: 
.sup.1 H NMR (CDCl.sub.3) .delta.2.61 (br s, 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 
.sup.13 C NMR (CDCl.sub.3) .delta.44.44, 50.74, 51.03, 51.85, 52.51, 60.28, 
115.61, 122.27, 137.24, 156.61. 
EXAMPLE G 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
ediethylphosphonate 
A mixture of 1 g (4.8 mmol) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene (prepared by 
the procedure of Example C), 4.8 g (28.8 mmol) of triethyl phosphite and 
864 mg (28.8 mmol) of paraformaldehyde was heated at 90.degree. 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 the organic eluent, the 
desired product was isolated as a colorless oil, 2.0 g (64%) and 
characterized by: 
.sup.1 NMR (CDCl.sub.3) .delta.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 
.sup.13 C NMR (CDCl.sub.3) .delta.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 
.sup.31 p NMR (CDCl.sub.3) .delta.24.92 (s, 2P), 24.97 (s, 1P). 
EXAMPLE H 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
edi(n-propyl)phosphonate 
To 3 mL of a chloroform/dioxane solution (1:1) was added 100 mg (0.48 mmol) 
of 3,6,9,15-tetraazabicyclo9.3.1!pentadeca-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.degree. C. with stirring for 1 hr. The resulting homogenous solution 
was concentrated in vacuo to give a viscous oil which was chromatographed 
on a neutral alumina column, eluting with chloroform. After concentration 
of the organic eluent, the desired product was isolated as a colorless 
oil, 320 mg (90%) and characterized by: 
.sup.1 H NMR (CDCl.sub.3) .delta.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 
.sup.13 C NMR (CDCl.sub.3) .delta.9.96, 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, 
57.16; and 
.sup.31 p NMR (CDCl.sub.3) .delta.24.98 (3P). 
EXAMPLE I 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
edi(n-butyl)phosphonate 
A mixture of 500 mg (2.4 mmol) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene (prepared by 
the procedure of Example C), 2.0 g (8 mmol) of tributyl phosphite and 240 
mg (8 mmol) of paraformaldehyde was heated at 100.degree. 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, eluting with 
chloroform. After concentration of the organic eluent, the desired product 
was isolated as a colorless oil, 1.25 g (65%) and characterized by: 
.sup.1 H NMR (CDCl.sub.3) .delta.0.84 (m, 18H), 1.27 (m, 12H), 1.58 (m, 
12H), 2.57 (m, 2H), 3.01(d, 6H), 3.99 (m, 12H), 7.12 (d, 2H), 7.54 (t, 
1H); and 
.sup.13 C NMR (CDCl.sub.3) .delta.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, 60.71, 60.78, 
65.38, 65.48, 65.58, 122.46, 136.96, 157.14; and 
.sup.31 P NMR (CDCl.sub.3) .delta.24.88 (2P), 24.93 (1P). 
EXAMPLE J 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3(4-nitrophen 
yl)methyl acetate! 
To a solution of 2.5 mL of chloroform which was rapidly stirred and 200 mg 
(0.97 mmol) of 3,6,9,15-tetraazabicyclo9.3.1!pentadeca-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, eluting with 
chloroform/methanol/ammonium hydroxide (16:4:1). After concentration of 
the organic eluent, the desired product was isolated as a light yellow 
solid, 250 mg (64%) and characterized by: 
.sup.13 C NMR (CDCl.sub.3) .delta.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. 
FINAL PRODUCTS 
EXAMPLE 1 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-trimethy 
lenephosphonic acid (PCTMP) 
A mixture of 2.06 g (10 mmol) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-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.degree. 
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 product 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.degree. C. and further characterized by: 
.sup.1 H NMR (D.sub.2 O) .delta.2.83 (m, 6H), 3.46 (m, 10H), 7.28 (d, 2H), 
7.78 (t, 1H); and 
.sup.13 C NMR (D.sub.2 O) .delta.53.61, 53.81, 55.27, 57.93, 62.20, 125.48, 
143.08, 152.31; and 
.sup.31 F NMR (D.sub.2 O) .delta.8.12 (2P), 19.81 (1P). 
EXAMPLE 2 
Preparation of the complex of .sup.153 
Sm-3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-trime 
thylenephosphonic acid (.sup.153 Sm-PCTMP) 
A solution of the ligand of Example 1 was prepared by dissolving 3.8 mg of 
ligand/0.517 mL of deionized water (pH=2). A 1:1 ligand/metal complex was 
then prepared by combining 40 .mu.l of the ligand solution with 2 mL of 
aqueous SmCl.sub.3.H.sub.2 O (3.times.10.sup.-4 M in 0.01N HCl) containing 
tracer .sup.153 SmCl.sub.3. After thorough mixing, the percent metal as a 
complex was determined by passing a sample of the complex solution through 
a Sephadex.TM. column, eluting with 4:1 saline (0.85% NaCl/NH.sub.4 OH), 
and collecting 2.times.3 mL fractions. The amount of 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. By this 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. 
EXAMPLE 3 
Preparation of 3,9-diacetic acid-6-(methylenephosphonic 
acid)-3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene (PC2A1P) 
A concentrated hydrochloric acid solution (37%, 5 mL) of 
3,9-bis(methylenenitrile)-6-(methylenedimethylphosphonate)-3,6,9,15-tetraa 
zabicyclo9.3.1!pentadeca-1(15),11,13-triene (prepared in Example F), 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.times.10 mL) to 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: 
.sup.1 H NMR (D.sub.2 O) .delta.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 
.sup.13 C NMR (D.sub.2 O) .delta.52.35, 54.04, 57.02, 59.24, 62.26, 125.52, 
143.64, 152.36, 171.54; and 
.sup.31 P NMR (D.sub.2 O) .delta.20.03. 
EXAMPLE 4 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
eethylphosphonate tris(potassium salt) (PMEHE) 
To an aqueous 0.1N potassium hydroxide solution (2 mL) was added 250 mg 
(0.38 mmol) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
ediethylphosphonate (prepared by the procedure of Example G). The solution 
was heated at 90.degree. 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: 
.sup.13 C NMR (D.sub.2 O) .delta.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 
.sup.31 NMR (D.sub.2 O) .delta.20.58 (s, 2P), 20.78 (s, 1P). 
EXAMPLE 5 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
e(n-propyl)phosphonate tris(potassium salt) (PMPHE) 
To an aqueous solution of potassium hydroxide (0.5 mL of 1N/dioxane (0.5 
mL) was added 81 mg (0.108 mmol) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
edi(n-propyl)phosphate (prepared by the procedure of Example H). 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: 
.sup.31 P NMR .delta.20.49 (s, 3P). 
EXAMPLE 6 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
e(n-butyl)phosphonate tris(potassium salt) (PMBHE) 
To an aqueous solution of 35 mL of 1N potassium hydroxide was added 3.21 g 
(3.88 mmol) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3,6,9-methylen 
edi(n-butyl)phosphate (prepared by the procedure of Example I). 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 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: 
.sup.1 H NMR (D.sub.2 O) .delta.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 
.sup.13 C NMR (D.sub.2 O) .delta.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 
.sup.31 P NMR .delta.9.73 (s, 2P), 21.00 (s, 1P). 
EXAMPLE 7 
Preparation of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3(4-nitrophen 
yl)methyl acetate!-6,9-methylenediethylphosphonate 
A solution of 250 mg (0.62 mmol) of 
3,6,9,15-tetraazabicyclo9.3.1!pentadeca-1(15),11,13-triene-3(4-nitrophen 
yl)methyl acetate!(prepared by the procedure of Example J), 624 mg (3.7 
mmol) of triethyl phosphite, and 111 mg (3.7 mmol) of paraformaldehyde was 
stirred at 100.degree. C. for 1 hr. The resulting homogeneous solution was 
concentrated in vacuo to give a viscous oil. The oil was dissolved in 10 
mL of chloroform and washed with water (3.times.5 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: 
.sup.31 P NMR .delta.24.67 (s, 2P), 24.88 (s, 1P). 
BIODISTRIBUTION 
General Procedure 
Sprague Dawley rats were allowed to acclimate for five days then injected 
with 100 .mu.L of the complex solution via a tail vein. The rats weighed 
between 150 and 200 g at the time of injection. After 30 min. the rats 
were killed by cervical dislocation and dissected. The amount of 
radioactivity in each tissue was determined by counting in a NaI 
scintillation counter coupled to a multichannel analyzer. The counts were 
compared to the counts in 100 .mu.L standards in order to determine the 
percentage of the dose in each tissue or organ. 
The percent dose in blood was estimated assuming blood to be 7% of the body 
weight. The percent dose in bone was estimated by multiplying the percent 
dose in the femur by 25. The percent dose in muscle was estimated assuming 
muscle to be 43% of the body weight. 
In addition to organ biodistribution, chelates of the compounds of Formula 
(I) were evaluated for efficiency of bone localization since phosphonates 
are known for their ability to bind to hydroxyapatite. 
EXAMPLE I 
The percent of the injected dose of complex of of Example 2 (.sup.153 
Sm-PCTMP) in several tissues are given in Table I. The numbers represent 
the average of a minimum of 3 rats per data point. 
TABLE I 
______________________________________ 
% INJECTED DOSE IN SEVERAL 
TISSUES FOR .sup.153 Sm-PCTMP 
Tissue 
Average 
______________________________________ 
Bone 34.87 
Liver 0.99 
Kidney 
1.42 
Spleen 
0.07 
Muscle 
4.77 
Blood 6.27 
______________________________________ 
EXAMPLE II 
The percent of the injected dose of complex of of Example 5 (.sup.153 
Sm-PMPHE) in several tissues are given in Table II. The numbers represent 
the average of a minimum of 3 rats per data point at 2 hours post 
injection. 
TABLE II 
______________________________________ 
% INJECTED DOSE 
.sup.153 Sm-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 0.12 
Brain 0.00 
Stomach 0.44 
Small Intestine 
10.71 
Large Intestine 
2.17 
______________________________________ 
EXAMPLE III 
The percent of the injected dose of complex of of Example 6 (.sup.153 
Sm-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. 
TABLE III 
______________________________________ 
% INJECTED DOSE 
.sup.153 Sm-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 of the injected dose of complex of of Example 3 (.sup.153 
Sm-PC.sub.2 A1) 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. 
TABLE IV 
______________________________________ 
% INJECTED DOSE 
.sup.153 Sm-PC2A1P (2 hours) 
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 
______________________________________ 
IMAGING EXPERIMENTS 
General Procedure 
Injectable solutions were first prepared (0.5M) by dissolving the 
appropriate amount of each complex in 2 mL of deionized water. The pH of 
the solutions were then adjusted to 7.4 using 1M HCl or NaOH as needed. 
The total Gd content of each solution was then determined by ICP analysis. 
An anesthetized Sprague Dawley rat was injected intramuscularly with one of 
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 with 
a 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. 
Other embodiments of the invention will be apparent to those skilled in the 
art from a consideration of this specification or practice of the 
invention 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.