CA2162136A1 - Bicyclopolyazamacrocyclophosphonic acids, their complexes and conjugates, for use as contrast agents, and processes for their preparation - Google Patents

Abstract

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. Processes for the preparation of the ligand, complex and conjugate are also disclosed.

Description

2 ~ 6 2 1 3 6 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 bicyclopolyazamacrocyclophosphonic 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 inthe following section.
Backqround MRI isa non-invasivediagnostictechniquewhich produceswell 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, 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 ti Ited out of al ignment with the magneti c 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 asspin-lattice (T1) and spin-spin (T2) relaxation and it isthese 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 ti mes. The greater the differences i n 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 i n this regard is related, i n part, to the num ber of unpa i red 30 electrons producing the magnetic moment [Magnetic Resonance Annual, 231 -266, Raven Press, NY (1985)]. It has also been shown that when a paramagnetic chelate of this type is administeredtoalivinganimal,itseffectontheT1 andT2Ofvarioustissuescanbedirectly 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 paramagneticchelatesbeadministeredtoanimalsinordertoincreasethediagnostic information obtained by M Rl [Frontiers of Biol. Energetics !, 752-759 (1978); J. Nucl. Med. 25, 506-513 (1984); Proc. of NMR Imaqinq Symp. (Oct. 26-27,1980); F. A. Cotton et al., Adv. Inorg.

Chem. 63~639 (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, ho~cvcr, the most useful 5 paramagnetic metal ions are gadolinium (Gd~3), iron (Fe~3), manganese (Mn 12) and (Mn ~3), and chromium (Crt3), 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 roleoftheorganicchelatingagent(alsoreferredtoasaligand)istorendertheparamagneticmetal non-toxic tothe 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 thatthe following summary, not intended to be exhaustive, is provided only as a review of this area and other compounds that are possibly similar in structure. U .S. Patent 4,899,755 discloses a method of alternating the proton NMR relaxation times in the liver or bile duct of an animal using Fe+3-ethylene-bis(2-hydroxyphenylglycine) complexes and its derivatives, and suggests among various other compounds the possible use of a pyridine macrocyclomethylenecarboxylic acid. U.S. Patent 4,880,008 (a CIP of U.S. Patent 4,899,755) discloses additional imagi ng 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 bifunctional macrocyclic phosphinic acid compounds. C.J.Broanetal.,J. Chem.Soc.,Chem. Commun., 1738-1739(1990)describe com pou nds that are triazabi cycl o com pou nds. I . K. Ad za m I i et al ., J. Med. Cl~em. 32, 139- 144 (1989) describes acyclic phosphonate derivatives of gadolinium complexes for NMR imaging At the present ti me, the only commercial contrast agents avai I able i n the U .S.A.
are the complex of gadolinium with diethylenetriaminepentaacetic acid (DTPA-Gd ~3 -MAG N EVIST'~ by Scheri ng AG) and a DO3A derivati ve [1,4,7-tris(carboxymethyl )- 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecanato]gadolinium(PROHANCE"' bySquibb).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. I n addition to MAGN EVIST~, Guerbet is commercially marketing a macrocyclic 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.
Surprisingly, it has now been found that various bicyclopolyazamacrocyclo-phosphonic acid ligands can be contrast agents. Furthermore, these ligands may have their chargemodified,i.e.bythestructureoftheligandandmetalselected,whichcaneffecttheirabilityto be more site specific. Specifically, the present invention is directed to novel ligands that are bicyclopolyazamacrocyclophosphonic acid compounds of the formula `~ 5 Q ,A

N ~
( I ) R-N N-R
-\~ ' R

wherein:

R = ~(C)n -T;
y where:
X and Y are independently H, OH, C,-C3 alkyl or COOH;
20 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, Cl-c18 alkyl, COOH, OH, 503H, --$--R4 ~

where: R1 is OH, Cl-C5 alkyl or --(Cl-C5 alkyl);
R4 is H, NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;
R2 is H orOH; with the proviso thatwhen R2 is OH, then the Rterm containing the R2 must have all X and Y equal to H;
with the provlso that at least one T must be P(O)RIOH, and with the proviso that when one T is ~) then one X or Y of that R term may be COOH and al I other X and Y terms of that R term m ust be H;
A is CH, N, C-Br, C-CI, C-oR3, C-ORa, N ' -Rs X-, C-C--C~ R4 R3 is H, C,-Cs alkyl, benzyl, or benzyl substituted with at least one R4;
R4 is defined as above;
Rs is Ct-C16 alkyl, benzyl, or benzyl substituted with at least one R4;
R8 j5 C1-CI6 alkylamino;
X- is Cl, Br', I or H3CCO2-;
Q and Z independently are CH, N, N +-Rs X-, C-CH2-oR3 or C-C(O)-R6;
Rs is defined as above;
R6 is --(C1-C3 alkyl), OH or NHR7;
R7 is C,-Cs alkyl or a biologically active material;
X~ is defined as above; or pharmaceutically-acceptable salts thereof;
with the proviso that:
a) when Q, A or Z is N or N ~-RsX-, then the other two groups must be CH;
b) when A is C-Br, C-CI, C-oR3 or C-OR8, then both Q and Z must be CH;
c) the sum of the R4, R7 and Rs terms, when present, may not exceed one; and d) only one of Q or Z can be C C(O)-R6 and when one of Q or Z is C-C(O)-R6, then A
must be CH.
When the above ligands of Formula (I) have at least two of the R terms T equal to PO3H2 [P(O)RIOH where R1 is OH] and the third T equal H, COOH or Cl-c18 alkyl; A, Q and Z are CH; n is 1; and X and Y independently are H or Cl-C3 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'OH, where R' is OH; n is 1; and X and Y are H. The use of these ligands is discussed and claimed in other copending applications.
When the above ligands of Formula (I) have:
intheRtermatleasttwoTequalP(O)R'OH,whereR'isOH,andintheotherR
term, r is COOH or P(O)R'OH, and n, Rl, X, Y, A, Q and Z are defined as above;
inatleastoneRtermTisP(O)RlOH,whereRlisOH,andintheothertwoRterms, T is COOH or P(O)R'OH, and n, Rl, X, Y, A, Q and Z are defined as above; or in the R term three T equal P(O)R1OH, where R' is Cl-C5 alkyl or -O-(C,-Cs alkyl), and n, R', X, Y, A, Q and Z are defined as above;
then the ligands are useful as contrast agents.

WO 94/267~4 2 1 ~ ~ 1 3 6 PCT/US93/04325 Particularly preferred are those ligands of Formula (I) where:
XandYareH;
n is l ; or A, Q and Z are CH.
Preferrably the ligands and complexes of Formula (I) do not have all three T equal to PO3H2 lP(O)RIOH where R1 is OH] when A, Q and Z are CH; although such complexes are useful as contrast agents or oral care agents. Thus the ligands and complexes of Formula (I) may have a proviso that not all T may be equal to PO3H2 [P(O)RIOH where R1 is OH] when A, Q
and Z are CH, unless used as a contrast agent or oral care agent.
BifunctionalligandsofFormula(l)aredesirabletopreparetheconjugatesofthis invention. Such ligands must have:
one R term where the T moiety is or ~ , where R2 and R4 are defined as above, especially where in the two R terms not containingan R4term, bothTtermsareP(O)R'OH,where R' isdefined asaboveor where in the two R terms not containing an R4term, one T term is a COOH and the other T term is P(O)R'OH, where Rt is defined as above; preferrably that moiety of the above T term where one of X or Y of that term is COOH; and also preferred are those ligands where n is 1 and/or the remaining X and Y termsare H ; or A is C-oR3 or C-OR8, where R3 and R8 are defi ned as above or C-C--C~ R4 where R4 is defined as above; or A is CH, and one of Q or Z is CH and the other is C-C(O)-R6, where R6 is defined as above;
especiallythose ligands where R6 is NHR7, where R7 is a biologically active material.
The ligands of Formula (I) may be complexed with various metal ions, such as gadolinium (Gd~3), iron (Fe~3), and manganese (Mn~2), with Gd~3 being preferred. The complexes so formed can be used by themselves or can be attached, by being covalently 35 bonded to a larger molecule such as a dextran, a polypeptide or a biologically active molecule, including an antibody or fragment thereof, and used for diagnostic purposes. Such conjugates and complexes are useful as contrast agents.

The complexes and conjugates of Formula (I) can be designed to provide a specific overall charge which advantageously influences the in vivo biolocalization and image contrast.
For example, when the metal ion is + 3 the following can be obtained:
(A) an overall chargeof -2 or more-when in three R terms T is P(O)R;OH, where R1 is OH, and n is 1; or in two R terms T is P(O)R1OH, where R1 is OH, in the third R term T is COOH, and n is 1 ; or in two R terms T is P(O)R1OH, where Rl is OH, in the third R term T is P(O)R'OH,where R1 j5 C1-C5 alkyl, and n is 1; or i n two R terms T is P(O)R'OH, where R1 is OH, i n the thi rd R term T is P(O) RIOH, where R1 is--(C1-C5 alkyl), and n is l; or (B) an overall charge of -l - when in one R term T is P(O)RiOH, where R1 is OH, and in the other two R terms T is P(O)R1OH, where R1 is -O-tC1-Cs alkyl), and n is 1; or in one R term T is P(O)R1OH, where R1 is OH, and in the other two R terms T is P(O)R1OH, where R1 j5 C1-C5 alkyl, and n is l; or in one R term T is P(O)R1OH, where R' is OH, and in the other two R terms T is COOH,andnisl;or (C) an overall neutral charge - when in the three R terms T is P(O)R1OH, where R1 is --(C1-C5 alkyl), and n is l; orin the three R terms T is P(O)R1OH, where R1 is Cl-Cs alkyl, and n is l; or (D) an overall charge of + l - when one of A, Q or Z is N ~-Rs X, where Rs and X are defined as above; and in one R
term, the T moiety is P(O)R1OH, where R1 is C1-C5 alkyl or -O-(CpCs alkyl); and in the other two R
terms, the T moiety is COOH or P(O)R1OH, where R' is C1-C5 alkyl, --(C1-C5 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 Formula (I) with other metal ions for diagnosis of disease30 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 desi red tissue. The advantage bei ng i ncreased contrast i n the areas of interest based upon tissue affi nity as opposed to contrast arisi ng from non-specific perf usion 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 wo 94/26754 2 ~ 6 2 1 ~ 6 PCT/US93/0432~
-, A

~1 N ~

14 N /~¦

complex. The overal I range of the charge of the complex is from -3 to + 1. For example, for a complex having 2 or more PO3H2 groups, the overall charge is highly negative and bone uptake is expected; whereas when the overall charge of the complex is 0 (thus neutral), the 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 20 macromolecu le can further i ncrease the contrast agent efficiency resulti ng i n i mproved 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 interactionswith blood proteins resulting in enhancement of relaxivity.
Additionally, the present contrast agents of Formula (I) which are neutral in charge are particularly preferred for forming the conjugates of this invention since undesirable ionic interactions between the chelate and protein are minimized which preserves the antibody immunoreactivity. Also the present neutral complexes reduce the osmolarity relative to DTPA-Gd '3, which may alleviate the discomfort of injection.
While not wishi ng 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 i n Form u la (I) are fu rther defi ned as fol I ows. "C,-C3 al kyl ", " C1-C5 alkyl~, ''Cl-c18 alkylN, include both straight and branched chain alkyl groups. An "animalN
includes a warmblooded mammal, preferably a human being.

216~136 ~ Biologically active materialN refers to a dextran, peptide, or molecules that have specific affinity for a receptor, or preferably antibodies or anti body fragments.
~ Antibodyn 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 using the term Nradioactive metal chelate/antibody conjugate" or "conj ugate~, the "anti body is meant to i ncl ude whole anti bod ies 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 111~NS-l9-9,1116-NS-3d, 703D4, 704Al, CC49, CC83 and B72.3 are deposited with the American Type Culture Collection, having the accession numbers ATCC HB 8û59, 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 Formula (I) complexed with a metal ion, where at least one metal atom is chelated or sequestered;
"conjugate~ referstoa metal ionchelatethatiscovalentlyattachedtoanantibodyorantibody fragment. Theterms "bifunctional coordinatorn, ~bifunctional chelating agentN and "functionalized chelantN are used i nterchangeably 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 bifunctional chelating agents described herein (represented by Formula 1) 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 1), 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 i nvention. Representative of those salts formed by standard reactions from both organi c and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, 35 lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutamic, gluconic acid, _-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 i norganic sources such as -ammonium or 1-deoxy-l-(methylamino)-D-glucitol, alkali metal ions, alkaline earth metal ions, and other similar ions. Particularly p.c~r~r.ed 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 processff are provided by the reaction schemes given below.
In Scheme l, the compounds of Formula (I) are prepared wherein X and Y = H, n = l (butwouldalsoapplyifn = 20r3withthecorrespondingchangeinthereagent), T = P03H2, and Q, A and Z = CH.

21 621 36:
WO 94/26754 PCT/US93/0432~

r \ ,, ~Z Z~ O
, r _ m ~Z~

Z u~ C~ ~ æ-- O
rz-~ Io ~
Lz~

~ c~ N
a~ r u U~ ~ Z N O

~ S ~ Z- ~ ~
O ~

O X

~ ~ m ~. ~

wo 94/26754 2 1 62 1 36 PCTtUS93tO4325 -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
=
o n -P-OH;
Rl where R' = -O-(C1-Cs alkyl); and Q, A and Z = CH.

V o V o O -- _ H

N ~ 5 \
~ Z_ _ O ~ ~ Z_~ V U
O ~ o/P
<~"Z Z~=O ~,Z Z~=O

N ~N=

~v aJ t v O~

V ~ V
O / O
u ~ a) <
~=~z~ ~Z \>$o =--~Z Z~ =o O ~= o ~ < ~- o o N -- O ` Y
o=~ ~ z _wo 94/26754 2 1 6 2 1 3 6 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
=

R

Rl where Rl = C1-C5 alkyl; and Q, A and Z = CH.

2l62l36 O 'I~ _ ~ U oo= ~/ _ O= ~ ~
\

iz~ ~Z_>
< \~=0 ~--O o\~

E~ ~ U r~
s U~

N
m cr m ~rl ~ =~--o o ~ C~

_wo 94/26754 ;~ 1 6 2 1 3 6 PCT/US93/04325 Scheme 4 prepares the compounds of Formula (I) wherei n X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change i n the reagent), T
=

- -P-OH;

whereR1 = -O-(C,-Csalkyl)orC,-Csalkyl;A = C-Br,andQandZ = CH.

m r~
m~=\z ~-m m ~ s u ~
ù m x O
s~ a r o ~ ~
,, ~< -- _ m m z_ Z w u~
u~
O J
~o m~z ~ rZz-E~

L _ U' ~
o Z E~
o ~

m ~ _, U

r--~

~i ~ ~ _ U
o~æ

\ir m _WO 94/26754 2 1 6 2 1 3 6 PCT/US93/04325 ,~ o '~ N
U O
\ I : -- t'`~ :I
~= O Q, ~ U~O
F ~ = o O ~~I /o ~ ~=0 P~ O ~ u :~ m u ~,/ O ~

U ~
O U O C H
_ O

v r ~ ~ ~ O E
um ~ z ~ ~J

s ,~ ;U~ '' m _ \O m u~ ~ V ~m ^\~
o \ :~ ~ V
~ ~C ~ ~ U -- o 1 /~ ( ` ~ ~

\_~ ~=o o ~/~o 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 correspondi ng change i n the reagent), T

-P-OH;
R

where R' = --(C1-C5 alkyl) or C1-C5 alkyl; A =

C-C-~S ;

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

_ WO 94/26754 2 1 6 2 1 3 6 PCT/US93/04325 iz_~

E

U, Z Vi r /~
vi ~-~Z Z~

~ v C~ ~ ~
U
i, il ~

WO 94/26754 2 1 6 2 1 3 6 PCT/USg3/04325 ~ H O

O O ~'~
o E E ~ o ~
<~

o o O

o ~ / m U~ ~ /

O _ /
U \ O ~, ~P~ = O -- ~ _ ~Z

'~ Z-- U
~ O X--- o~ ~ = o ",\m o -- E~ ~ ~\
fl ~ ~ o o \
o ~ W

r~

wo 94/26754 2 1 6 2 1 3 6 PCT/US93/04325 H
O ~ O

U ~ ~ H
O
~ O ~
C~ O ~ O
,< ~1 = O ~

r \ ~ ~ ~ ~ _ a t :~

~o=~

Scheme 6 preparesthe 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 R' = --(Cl-C5 alkyl) or Cl-C5 alkyl;
A = C-OR8, where R8 = C1-Csalkylamino; and QandZ = CH.

WO 94t26754 2 i 6 2 1 3 ~ PCT/USg3/04325 o Z mO

a o O
U~
O ~ o U ' U
- ~ ~U
Il o~

O V ,~ U U
~0 rZ-E~o E ~ o _ Z--E~

Z
lr u~ --O~z Z~ E~
V ~ Z_/

J.L o 5 E~

>
Z--uO mO o m ,<~= a~
O,V/
o~z ~ =o Z--_, /~= O ~

V
o o m ,< P~ = O
$ ~ z ~=o o Z
~4 O ~ = O ~ _ " ~ CJ o ~
~ o= ~ ,, Z

o ~ Z_~ O

o~z ~--m ~ Z_ ~
m o m Z\ ~
m ~ ~ z--o ~\ Z 7~
m ~ \~/ ~ _ ~ ZJ ~
m O ~
<~= O o Z~o~ ~V=

<~=o UO H
U
Z~ V
E ~~ o~o ~< ~ = O O

Z~ /=( )1/
o ~z ~ --o <
/~ /~= O
O ~ ~s3 ~) ._, WO 94/26754 2 1 6 ~ l 3 6 PCT/US93/04325 o o t'l 'O H ~ H

<P~= O _ C~

Z~o~ < ~ = o /0= ~ 7=

v m P~=o o C,~ o o ~ ~
E m o ~

m / u ,~ _ U
'` / = ~--O o ~ U

WO 94/26754 ~ 1 6~1 36 PCTIUS93/04325 Scheme 7 prepares the compounds of Formula (I) wherein X and Y = H, n = l (but would aiso apply if n = 2 or 3 with the corresponding change in the reagent), T

1l -P-OH

where R' = -OH, -O-(C,-Cs alkyl) or C,-Cs alkyl;
Z = C-C(O)-R6,where R6 = OH; and Qand A = CH.

5, a~
5~ Z--O E~ ' 5t ~z ~ ~ m 5Z~

Z Z L~
m ~

Z~
rz-~
L z _ E~ ~ \
Z ~
a U~ \
\

t~ ~Z ~ \

~U
o O = u 5 X

~i U ~, U~
Z

O ~ O O
O<P~=O -- mO <~=

o ~

O ~= O E

O O
O ~ , ~

~, / ~ o <
a ~ o= ~_ 5 ' <~ Z ~ , a U \~ < ~ = O o o O

m ~ u o O
a~ \ ~ O m ~
~Z ~ ~ ~ o <p, o ~ ~

Scheme 8 prepares the compounds of Formula (I) wherein X and Y = H, n = l (but would also apply if n = 2 or 3 with the correspondi ng change i n the reagent), T =
ll -P-OH;

where R' = -OH, -O-(C,-Cs alkyl) or Cl-C5 alkyl;
Z = C-CH2-OR3where R3 = benzyl; and QandA = CH.

WO 94/267~4 2 1 6 2 1 3 6 PCT/US93/04325 O ~

~ ~ =P~_ o (\ Z Z~

Z--) \ 1 ~ \ ~) O X ~

< ~0 <~ ~ ~ ~

E ~

= E

216213~

O ~ Ln ) O
~ ~V~

<
1 ~
~ o O U
:C ~

-o E < O
o ~ ~ e V~<~ Z~ ~ _ z ~ 2=o ~o )V
Z o < O
~= o ~
g o u P~ U
:C

Ln n ~WO 94/26754 2 1 6 2 1 3 6 PCT/USg3/04325 ~ 5:
U\o ~ ~ = O O

) 1/ o ~Z ~ =o ~P = o o ~ C ~ _ ~-~ < ~3'= E

) ~0 (\ Z ~ ~= O ,~
\V

< O
~= o U
~o ~ E~
U
O _ _ p, ~ ~:
O

r O
< 0 ) /
Z = O \~ E

~ \ uO
0/0= \\,~ _ v ~ O

O 0 ~7 0 O ~
C o ~ O

E C ~ '~ = O

o ,<~ = O ~
=

~ = O

O __~ O

r~ O ~
ly ~ o= I ~ Z

~: x ---~34-Scheme 9 prepares the compounds of Form u la (I) wherei n X and Y = H, n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T

1l -P-OH;

where R1 = -OH, --(Cl-C5 alkyl) or C1-C5 alkyl;
A = N or N-Rs; Rs = Cl-cl6 alkyl halide; and Q and Z = CH.

Vl ~

) u~
Z Z ~--~
~Z_) ~
U~
E~
rZ--~ o m ' --Z ~
L z _ E~ ~:
Z
$
cn ~U ~ ) Lr, Z Z ~--~
Z Z ~D \\ //
~ ~
Z_/
U ~

~_ X
N
U O
U~
Z --Z Z ~
\
U

~ WO 94/26754 2 1 6 2 1 3 6 PCT/US93/04325 5: 0 U U /
~ ~ U

=4 ~ ~ ~=o 1~

E ~ ~ ~ ON 5~ Z ~ Z ~ = O
v~ \ O O _ ~ _)\/ c \ :r: O ~; ~
U \ ~ ~ ~= O U
O \ ~a ~5 :C

~C _ 0 ~7 <P~ _ ,., O

~ )v ~ v ~

,<p~=O r` ~H \I=o ~D
,"_, 1',.,,;._~, O m m o= ~--o U ~ m u ~ o ~ / ~
r N ~ _ l--U ~ = O

O ~ O

c~ _ _ o ~ O=-- = ~

H z I X o o ~ 0 '~ m E~ ~ = o ~ ~ X ~,r ~ o/ o X , /~+~ Z ~- o ~ ~ ~ = O 0 ~ m ~ WO 94/26754 2 1 6 2 1 3 6 PCT/US93/04325 Scheme 10 prepares the compounds of Form u ia (I) wherei n X and Y = H, n = l (but would also apply if n = 2 or 3 with the corresponding change in the reagent), T

1l -P-OH;

where R7 = -OH, --(Cl-C5 alkyl) or Cl-C5 alkyl;
Q = N-Rs; Rs = C1-C16aikyl halide; and Aand Z = CH.

~ ~ X ~D

~ U~ --E~ I co rz-~o lx~

o Z--E~ ~ ~ Z
z a V~ r X
~Z ¢

Z--~ _ ~ô ~ X~ ~

Z-- :~

~z a~

~_WO 94/26754 21 621 36 PCT/US93/04325 ~ O < ~ H
'\ O ~ r Z~
~ o--~ E

X ~ \/ O

~= O U E~ -- U ~
~n ~ ~ Il~ O ~ ~ ~3 --U ~ ~_ ~
~: ~ <
~ o~ /~ O r \ G'~
U ~ ,3 U ~
m u / -- o ` ~ æ Z ~=
=~--o/,~, " \/\~ ) o=~ O ~ <Z--U o~
O ^ ~ 0~ ~=
cr^ ~~ U V
V C~ Y o E
O--P~ ' U
O ~ ,, U
~ U
U I X
X-- O
~ U
:~ m ~ N

X ~ X ~

~ O O 0 ~5 V ~
~C ~ O--Scheme l 1 prepares the compounds of Formula (I) wherei n X and Y = H, n - 1 (but would atso apply if n = 2 or 3 with the correspondi ng change i n the reagent), R

--P-OH;

where R' = -OH, --(C1-Cs al kyl) or C1-C5 al kyl;
Q = N or N-Rs, Rs = Cl-cl6 alkyl halide; and AandZ = CH.

~ WO 94126754 PCTIUS93/04325 U~ _ :C H
U\ 10 In <~=

r Z~ ~ O
~/ O O
Z~ )\/ ~

~ zJ ~ u~ O ~ ~
= U ,<P = O ~ -~
z_ ~
r , r~ O

O ~P~= O
E~ ~ U ~, U

=~_ o ~ 0~

/

/ m æ ~--:C ~ P' ~

. ~

O X
m -= o ~ ~ ~ O
~=0 ~ o/o o '' m ~0 ~, O
X 'u \~ ¢ l~

i ' ` ~ ' ~ U X ~ X <~
U~ (~ O ' N N
)-I O _ ~ ' ~I r O I O

O~ O

O= ~ ~t O O

U

p~
~C~
~D ~ ~

~WO 94/26754 2 1 6 2 1 3 6 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-substitutedsulfonatederivative,which isthenconvertedtotheco"es~,onding nitrile.
Sebsequent phosphonomethylation and hydrolysis yields the desi red product.

Scheme 12 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 position has T =

-P-OH;
R

where R1 = -OH or -O-(C,-Cs alkyl); and the other two R terms have T = COOH; andA, Qand Z = CH.

_ ~ _ O

Z ~ ~ C

u' a~
~C
O ~
o o O
r~ =~v æ-- ~:
U
<~æ z~ _ ~ Z _>

m~
æ~

æ
~ ~ _ <~Z Z_~ ~

Z _ ~WO 94/26754 2 1 6 2 1 3 6 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;
Rl where R1 = OH or-O-(C,-Cs alkyl); and the other R term atthe 9 position has T = COOH;
and A,QandZ = CH.

WO 94/26754 ~ 1 6 ~ 1 ~ 6 PCT/US93/04325 ~ H ~
O O
rU ~rU
æ_~ E~ æ~ O

i ~ _ 5 .- ~ H

v Ln O E
o W U
N ~ ~ 4 ~ _ ~
o _ ~3 U o P~ ~ ~ O
O _ O ~
r u _ o= ~
s r æ--O z_ æ~ æ

~ C~ ~
o Z

r æ~

'~Z Z--Scheme 14 prepares the compounds of Formula (I) wherein X and Y = H, n = 1 (butwould also apply if n = 2 or 3 with the corresponding change in the reagent), R
terms atthe 3 and 9 positions have T =
o -P-OH;

where R1 = -OH or -O-(C,-Cs alkyl); and the other R term at the 6 position has T =
COOH; and A,QandZ = CH.

WO 94/267542 l 6 2 1 3 ~ PCT/US93/04325 H H
--~ = 0 ~ ~ \ ~

a ~ ~ c L 11, E~
~ ~ O u m ~ o o \

V
~ o -u m U~ U
o -- o ~U ~ :~
m m o= P~
\/

~ Z~
~ \ ~
~ Z _~ _ WO 94/26754 2 1 6 2 1 3 6 PCT/US93/0432~

Scheme 15 prepares the compounds of Formula (I) wherein n = l (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 T =
C

-P-OH;

where R' = -OH or -O-(C,-Cs alkyl); and X and Y = H;
the R term at the 6 position has T =

~R4 where R4 = NO2or NH2; and one of X or Y = H and the other = COOH; and A, Qand Z = CH.

C)~ o \~ 0~
+ ~Z-~

> U

ù

Z--~ 5n~
~Z ~ ~0 m ~2 ,~ _ ~ ~ O -- O
. ~ ~
~ ~ ~ E
:r: 2 o ~4 Z-~ ~

_l ~ >~o~ ~
E ~ Z--U~ , ~
o O

O ~
~C_) O ~ _ ~=0 Z

Z~ E

~Z Z ~U --O
~ ~ r '7~

~ P~ = O U
O ~\ ~5 O O

--O Z

r i z ~ ~

V ~ O
O ~=0 0 O O
O O ~ ~
~ U I ~

o -_ WO 94/26754 Scheme 16 prepares the compounds of Formula (I) wherei n 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 =

-P-OH;

where R1 = -OH r-O-(Cl-Cs alkyl); and X and Y = H;
the R term at the 9 position has T =

~R4 where R4 = NO2 or NH2; and one of X or Y = H and the other = COOH, A,QandZ = CH.

N

~/~ 6 Z-~

_>
O o 1, U
o _I N

S

U~ H

m ~ ~ E

<~i Z~ '-~
Z~ ~ _ O U
N t~5 -/-=\ ~C --oN ~ E
,-- Z----o ~ ~ O ~
<~Z Z P~- O

V O

~=0 ~\
O O

V N

~D O

E ~N ~_~

~ ~ o~

U ~ ~ > O

,_J O O
o Scheme 17 prepares the compounds of Formula (I) wherei n n = l (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;
R
whereR1 = -OH;andXandY = H;
theRterm atthe3and 9positionshaveT = COOH; and A,QandZ = CH.

r~

Z /~ ~ O
<~z z--m r I--~ \m \ m~ ~
i z_> r L" /-Z--o ~ ~=< >
E Z ~ <\ z ~, o ~ >_ C~ E
o ~ m ~

z _> ~
m _ <~z z--m ~r m In the above Schemes, the general process d i scri ption i l l ustrates speci fic steps that may be used to accomplish a desired reaction step. The general description of these process steps follows.
The synthetic Scheme 1 begi ns with a halogenati on of commercial Iy avai labl e bis-pyridyl alcohol (1) using thionyl chloride. Similar procedures for converting an alcohol to an el ectrophi I i c su bstrate, such as treatment with tol uenesu I fonyl chlori de, H B r or H Cl, shou I d a I so result in a similarily reactive productwhich would work well in subsequent ring closure reactions. Macrocyclization proceduresare numerousin 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. Red uctive detosylation procedures are also well known i n the literature and can be adapted to the present reaction sequence. Phosphonomethylation to obtai n the tris-aminophosphoni c acid derative (5, PCTMP) was conducted u nder 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 nucleophi I ic species (see Scheme 2) . Dependi ng u pon the reactivity of the ami ne, these reactions may be conducted at a temperature between about -10 to about 1 00C. In addition, trialkylphosphites can be employed under similar Mannich conditions to give the phosphonate ester via oxidation of phosphorous (Ill) to phosphorous (V) with simultaneous expulsion of one mole of alcohol (Arbuzov reaction). These reactions can be conducted with or without the presenceofasolvent. Whenalcoholsareemployedasthesolventforeitherdialkylortrialkyl phosphite reactions, it is beneficial to use the alcohol from which the corresponding phosphonateesterisderived inordertoavoid alternativeproductsarisingfrom transesterification. Esters of this type are also prepared via N-alkylation of a-halo-dial kyl phosphonates i n solvents such as acetonitrile, chloroform, dimethylformamide, tetrahydrofuranor1,4-dioxanewithorwithouttheadditionofanon-nucleophilicbasesuchas potassium carbonate at room temperature or above. The resulting perester intermediate is then readily hydrolyzed under basic conditions (aqueous hydroxide, pH = 8-14, 30-110C) to give the corresponding half-acid derivative.
lnscheme3~macrocyclicmethylphosphinicacids(1oand 11)arepreparedunder conditions similarto 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 WO 94/26754 2 1 6 2 1 3 6 PCT/US93/0432~
phosphinate ester is then hydrolyzed under acid (6N HCI, 80- 100C) or basic (stoichiometric quantitiesofbase,40-100C)conditionstogivethecorrespondingmethylphosphonicacid.
Alternatively,themethoddevisedbyA.D.Sherryetal.(lnorg.Chem.,submitted 1991)usingethylphosphonic acid generated insitu 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-membered tertaazamacrocycle. Thus, chelidamic acid (Sigma Chemical Company; 12) can be converted tothe bis-halomethyl derivative (13) having appropriate substitution atthe pyridyl 4-position. Transformations leading tothis intermediate are general 10 i n 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 100Cwith 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 lead i ng to phosphonate half-acids and phosphi nate functional ity 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 descri bed i n Scheme 5. Thus, organometallic Pd(li) 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 uti I ize anhydrous conditions with triethylami ne as sol vent and at reaction temperature between about 10 to about 30C for optimum yields. The identical product can also be obtained using Cu(l) phenylacetylide i n anhydrous pyridine at a temperature between about 80 to about 110C. In addition, standard anionic alkylation 25 procedures can be employed to affect substitution on the pyridine nucleus with, for example, sodioalkoxides in DMF or dioxane at from about 80 to about 100C 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 35 butyll ithium at temperatures between from about -30 to about 80C. 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 tothe 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 macrocycl ic 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 ringwithin the macrocyclic structure as illusatrated in Scheme 7. Newkome et al. lTetrahedron 39(12),2001-2008(1983)]haspreviouslydescribedthesynthesisofethyl2,6-halomethylnicotinate (45) which serves as the inital starting material in this synthetic route.
Thus, the tris-tosylated macrocycle intermediate (46) can be detosylated under acidic conditions(HBr/AcOH,25-115C)withsimultaneoushydrolysistoyieldthenicotinicacid derivative (48), or reduction of the ester i n refl uxi ng ethanol prior to detosylation will resu It i n 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 20 catalytic hydrogenation conditions (58).
MacrocyclicderivativescanaisobepreparedasinSchemes12-14whereboth carboxylate and phosphonate chelating fuctionalities are present in the same molecule. Thus, varying degreesof carboxylatefuctionalitycan be introduced undertypical aqueousalkylation procedures using bromoacetic acid. Following this step, the remaining amines can be phos-25 phonomethylated by procedures discussed in previous Schemes using formaldehyde andphosphorousacid,dialkyl phosphonatesortrialkyl phosphites.
Schemes 15 and 16 del i neate a synthetic approach which i ntroduces an aromati c nitrobenzyl substitutent 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 30 temperature using a non-nucleophilic base such as potassium carbonate. Additional functionalization of the remaining nitrogen positions is then performed by methodsand conditions descri bed i n previous Schemes. After the i ntroduction of the desi red chelati ng moieties, the nitro group is reduced using platinum oxide and hydrogen i n water. In this form, the chelating agent is compatible with conjugation techniques which will enable attachment 35 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 ~o 94/26754 2 1 6 2 1 3 6 PCT/US93/04325 macrocyclic compound where positions 3 and 9 are the sodium salt of methanesulfonic acid (119). Thesulfonicacidgroupisthendisplacedusingsodiumcyanidetoformthe correspondingcyanomethanederivative(120). Thecyanogroupishydrolyzedtothecarboxylic acid either: simultaneousiy with the addition of phosphorous acid and formaldehyde; or by 5 sequential reaction with a derivative of phosphorous acid and formaldehyde to form the phosphonicacidatthe6position(121),followedbyacid hydrolysis,atanelevated 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 10 preformed by the methods discussed above The metal ions used to form the complexes of this invention are Gd ' 3, Mn ~2, Fe ~3 and available commercially, e.g. from Aldrich Chemical Company. The anion present is halide, preferrably chloride, or salt free (metal oxide).
A "paramagnetic nuclide" of this invention means a metal ion which displays spinangular 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 beari ng the unpaired electron and, to a lesser extent, upon the envi ronment 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 ' Z), with Gd +3 being preferred.
The complexes are prepared by methodswell 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 25 the metal ion under aqueous conditions at a pH from S to 7. The complex formed is by a chemical bond and results in a stable paramagnetic nuclide com position, 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 molarratioofatleastabout 1:1,preferablyfrom 1:1 to3:1,morepreferablyfrom 1:1 to 1.5:1.
30 Alargeexcessofligandisundesirablesinceuncomplexedligandmaybetoxictotheanimalor 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, Kohler and Milstein [Nature,256,495-497 (1975); and Eur. J. Immunol.,6, 511 -519 (1976)]. Such antibodies normal Iy have a highly specific reactivity. In the antibody targeted conjugates, antibodies directed against any desired antigen or hapten may be used. Preferably theantibodieswhich are used inthe conjugatesare monoclonal antibodies, orfragments thereof having high specificity for a desired epitope(s). Antibodies used in the presentinvention 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 5 antibodiesorantibodyfragramentsare 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 antigenscanbefoundinU.S.Patent4,193,983. Theconjugatesofthepresentinventionare 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 adj uvants, 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.
20 The ligands of Formula (I) having in at leasttwo Rterms T equal to P(O)RIOH may be used for metal ion control as scale inhibitors. Some of thffe 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 used in the following examples are defined as fol lows:
LC = liquid chromatrography, purifications were carried out at low pressure using Dionex 2010i system fitted with a hand-packed Q-Sepharose'~ anion exchange column (23 x 2 cm).
DMF = dimethylforamide.
AcOH = aceticacid.
ICP = inductively coupled plasma.
g = gram(s).
mg = milligrams.
kg = kilogram(s).
mL = milliliter(s).
L = microliter(s).

21 62~ ~
--WO 94/267~4 PCT/US93/04325 pH StabilitY General Procedure A stock ls9GdC13 (or l53SmCI3) sol ution was prepared by addi ng 2 IlL of 3x 10-~M
ls9GdCI3in0.1NHClto2mLofa3x10~MGdCI3carriersolution. Appropriateligandsolutions were then prepared in deionized water. The 1: 11igand/metal complexes were then prepared 5 by combi ning the I igands (dissolved i n 100-500 IlL of dei oni zed water) with 2 m L of the stock ls9GdCI3solution,followedbythroughmixingtogiveanacidicsolution(pH = 2). ThepHofthe solution was then raised to 7.0 using 0.1 N NaOH. The percent metal as a complex was then determi ned by passi ng a sample of the complex sol ution through a Sephadex '~ G-50 col umn, eluting with 4: 1 saline (85% NaCI/NH4OH) and collecting 2 x 3 mL fractions. The amount of 10 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 byadjusting the pH of an aliquot of the complex solution using 1M NaOH or lM HCI 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 biodistri bution of the I igands 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 9 (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 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 NaHCO3, filtered and dried to yield 23.1 9 (71.5%) of the titled product as an off-white crystalline solid, mp 74.5-75.5C, and further 25 characterized by:
H NMR (CDC13) 4.88 (s, 4H),7.25-7.95 (m,3H).
Exam,ole B
Preparation of 3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-30 triene A DMF solution (92 mL) of 6.9 9 (11.4 mmol) of 1,4,7-tris(p-tolylsulfonyl)diethylenetriamine disodi um salt was stirred and heated to 100C under nitrogen.
To the solution was added dropwise over 45 min 2 9 (11.4 mmol) of 2,6-bis(chloromethyl)pyridine (prepared by the procedure of Example A) in 37 mL of DMF. When 35 the addition was completed the reaction mixture was stirred at 40C 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 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 9 (86%), mp 168-170C dec. and further characterized by:

H NMR (CDC13) 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,1 H); and 5 ~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[9.3.11pentadeca-1(15),11,13-triene.
AsolutionofHBrandAcOHwaspreparedbymixing48% HBrandglacialAcOHin 10 a64:35 ratio. To 112 mLofthe HBr/AcOH mixturewasadded 5.59 (8.2 mmol)of 3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(prepared bythe 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 forrned which was filtered, washed with diethyl ether, and dried in vacuo. The dry tetrahydrobromide salt was thendissolvedin10mLofwater,adjustedtopH9.5withNaOH(50%w/w)andcontinuously 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 20 room temperature to yield 1.2 9 (71 %) of the title product, mp 86-88C and further characterized by:
1H NMR (CDC13) - ~ 2.21 (m,4H),2.59 (m,4H),3.06 (s,3H),3.85 (s, 4H),6.89 (d, 2H), 7.44 (t, l H); and 25 ~48.73,49.01,53.63,119.67,136.29,159.54.
Example D
Prepa rati on of 3,6,9,15-tetraazabi cycl o[9.3. l ] pentadeca- 1 (15),11,13-tri ene-3,9-dimethylenesulfonic acid.
A slurry of 500 mg (2.4 mmol) of 3,6,9,15-tetraazabicyclo[9.3 11pentadeca-30 1(15),11,13-triene (prepared bythe procedure of Example C) was stirred in 6 mL of water and the pH adjusted to 3 using 6M HCI. To the mixture was added 682 mg (5. l 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.

21621~

Example E
Preparation of 3,6,9,15-tetraazabicyclol9.3.1]pentadeca-1(15),11,13-triene-3,9-dimethylenenitrile.
Tothe reaction mixture containing 3,6,9,15-tetraazabicyclol9.3.1]pentadeca-1(15),11,13-triene-3,9-dimethylenesulfonic acid from Example Dwasadded 47 mg (9.6 mmol) of sodium cyanide. The reaction mixture was stirred at room temperature for 24 hrs. 13C NMR
i ndicated that transformation to the bis-nitri le was complete. The reacti on m i xture 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 cyclohexane, 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-tetraazabicyclo~9.3.1]pentadeca-1(15),11,13-triene (PC2S).
An aqueoussolution (10.0 mL) of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (prepared by the procedure of Example C), 1.03 9 (5.0 mmol) was added with 0.5 mL of concentrated HCI and stirred for 10 min to ensure complete dissolution. The resulting solution had a pH of 8.6. Tothe solution wasthen added 1.37 9 (10.2 mmol) of HOCH2SO3Na with 5 mL of deionized water. The solution was heated at 60C for 10 min and the pH dropped 20 to5.6. Aftercooling,thepHwasadjustedto9.0with lMaqueoussodium hydroxide,followed by Iyophilization to give the desired product as a white solid in a quantative yield and characterized by:
ZH NMR (D20) ~ 2.87 (t, 4H), 3.18 (t, 4H), 3.85 (s, 4H), 4.11 (s, 4H), 7.03 (d,2H), 7.55 (t,1 H); and 25 13C NMR (D20) ~48.52, 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[9.3.1]pentadeca-1(15),11,13-triene.
Toan aqueoussolution,10.0 mL, of 3,9-bis(sodium methylenesulfonate)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (prepared bythe procedure of Example F), 2.26 9 (5 mmol), was added 0.6 9 (12 24 mmol) of sodium cyanide. The mixture was stirrecl for 3 hrsatroomtemperature. ThepHofthereactionmixturewasaboutlO. ThepHwasadjusted 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 9 (71 %) and characterized by:

WO 94/267~4 2 1 6 2 1 3 6 PCT/US93/04325 'H NMR (CDCI3) 2.03 (br s, 4H),2.64 (m,4H), 3.82 (s, 4H),3.90 (s, 4H),7.14 (d, 2H),7.62 (t,1 H); and '3C NMR (CDCi3) ~ 46.08, 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-tetraazabicycl o[9.3.1] pentadeca- 1 (15),11,13-tri ene-3,9-d i methyleneni tri I e.
3,9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclo[9.3.11pentadeca-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 ThemixturewasgentlystirredforlOmintoobtainaslurry,thenheated to 90C for 1 hr. After the excess reagents and byproducts were removed in vacuo (1 hr at 125C/0.01 mmHg), he resulting dark brown residue was dissolved in 20 m L of chloroform and washed with deionized water (5 x 15 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, andtheexcesssolventsevaporated invacuotogivethedesired productasayellowwaxysolid,168mg(41%)andcharacterizedby:
'H NMR (CDCI3) 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, 1 H); and 20 13C NMR (CDCI3) 44.44, 50.74, 51.03, 51.85, 52.51,60.28,115.61,122.27,137.24,156.61.
Example I
Preparationof3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylenediethyl phosphonate.
Amixtureof 1 g(4.8mmol)of3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(prepared bytheprocedureof ExampleC),4.8g(28.8mmol)oftriethyl phosphite and 864 mg (28.8 mmol) of paraformaldehyde was heated at 90C 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 9 (64%) and characterized by:
lH NMR (CDCI3) â 1.23 (m,18H),2.77 (m,12H), 3.04 (d, 6H), 4 13 (m,12H~,7 17 (d, 2H), 7.60 (t,1 H); and 3C NMR (CDC13) 35 ~ 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~P NMR
24.92 (s, 2P),24.97 (s,1 P).

21 62l 36 Example J
Preparation of 3,6,9,15-tetraazabicyclo[9.3.11pentadeca-1(15),11,13-triene-3,6,9-methylenedi(n-propyl)phosphonate.
To3mLofachloroform/dioxanesolution(l:l)wasadded lOOmg(0.48mmol)of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (prepared bythe 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 90C 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 el uent, the desi red prod uct was isol ated as a colorless oi I, 320 mg (90% ) a nd characterized by:
'H NMR (CDC13) 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, 1 H);
and CNMR (CDC13) 9.96, 23.73, 4g.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 31p NMR
~ 24.98 (3P) 20 Example K
Preparation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylenedi(n-butyl)phosphonate.
A mixture of 500 mg (2.4 mmol) of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1 (15),11,13-triene (prepared by the proced ure of Example C),2.0 g (8 mmol) of tri butyl 25 phosphite and 240 mg (8 mmol) of paraformaldehyde was heated at 100C 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, l .25 9 (65%) and characterized by:
30 1 H N M R (CDCI 3) 0.84 (m,18H),1.27 (m,12H),1.58 (m, 12H),2.57 (m, 12H), 3.01 (d, 6H), 3.99 (m,12H), 7.12 (d, 2H), 7.54 (t, 1 H); and 3C NMR (CDC13) ~ 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'P NMR
24.88 (2P), 24.93 (1 P).

- WO 94/267~4 2 1 6 2 l 3 6 PCTIUS93/0432~
Example L
Preparation of 3,6,9,15-tetraazabicyclo[9.3.11pentadeca-1(15),11,13-triene-3[(4-nitrophenyl)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-tetraazabicyclo[9.3.1]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 concer,lrdled in vacuo to give a semi-solid which was chromatographed on a silica gel column, eluting with chloroform/methanol/ammonium 1 o hyd roxide (16: 4: 1). After concentrati on of the organi c el uent, the desi red prod u ct was i sol ated as a light yellow solid, 250 mg (64%) and characterized by:
3C NMR (CDC13) ~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-tetraazabicyclo[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-tetraazabicyclo[9.3 1]pentadeca-20 1(15),11,13-triene(preparedbytheprocedureofExampleC),11.3g(138mmol)ofphosphoric acid and 15 9 (152 mmol) of concentrated HCI was heated to gentle reflux (103 C) with constantstirringfollowed bythedropwiseaddition(2mUmin)of 12.2g(150mmol,15mL)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 product was then purified by LC anion exchange chromatography (0-30% formic acid, 3 mUmin, 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 275-280C and further characterized by:
'H NMR(D20) ~ 2.83 (m, 6H), 3.46 (m,1 OH), 7.28 (d,2H), 7.78 (t,1 H); and 30 i3C NMR
53 61, 53 81, 55.27, 57.93, 62.20,125.48,143.08,152.31; and 3lp NMR
~8.12(2P),19.81 (1P) Example 2 35 Preparationofthecomplexof1s3Sm-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-trimethylenephosphonic acid ('s3Sm-PCTMP) A solution of the ligand of Example 1 was prepared by dissolvi ng 3.8 mg of I igand/0.517 m L of deionized water (pH = 2). A 1: 11 i gand/metal complex was then prepared by combining 40 lul of the ligand solution with 2 mL of aqueous SmCI3-H20 (3x104M in 0.01 N HCI) containing tracer 153SmCI3. After thorough mixing, the percent metal as a complex was determined by passing a sample of the complex solution through a Sephadex'~ column, eluting with 4: 1 saline (0.85% NaCI/NH4OH), and collecting 2 x 3 mL fractions. The amount of 5 radioactivityinthecombinedelutionswasthencomparedwiththatleftontheresin. Underthese 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.
10 EXample 3 Preparation of 3,9-diacetic acid-6-(methylenephosphonic acid)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (PC2AlP).
A concentrated hydrocholric acid solution (37%, 5 mL) of 3,9-bis(methylene-nitrile)-6-(methylenedimethylphosphonate)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-15 1(15),11,13-triene(preparedinExampleH), 168mg(1.0mmol)washeatedatrefluxfor16hrs.
After cooling, the solution was evaporated to dryness, followed by coevaporation with deionizedwater(2x 10mL)toremovetheexcesshydrochloricacid. Thefilal productwas isolated as a dark brown solid upon Iyphilization of the concentrated queous solution and characterized by:
20 H NMR (D2O) 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,1 H); and 13C NMR (D2O) 52.35, 54.04, 57.02, 59.24, 62.26, 125.52,143.64, 152.36,171.54; and 31p NMR (D2O) 25 ~ 20 03 Examole 4 Preparation of 3,6,9,15-tetraazabicyclol9.3.1]pentadeca-1 (15),11,13-triene-3,6,9-methyleneethylphosphonate tris(potassium salt) (PMEHE).
To an aqueous 0.1 N potassium hydroxide solution (2 mL) was added 250 mg 30 (0.38mmol)of3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylene-diethyl phosphonate (prepared by the procedure of Example 1). The solution was heated at 90C 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:
13C NMR (D2O) 35 ~ 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 31p NMR
20.58 (s,2P), 20.78 (s, 1 P).

wo 94/26754 2 i 6 2 1 3 6 PCT/US93/04325 Example 5 Preparation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylene(n-propyl)phosphonate tris(potassium salt) (PMPHE).
To an aqueous sol ution of potassi um hydroxide (0.5 mL of 1 N/d i oxane (0.5 m L) 5 was added 81 mg (0.108 mmol) of 3,6,9,15-tetraazabicyclo[9.3.1] pentadeca- 1 (15),11,13-triene-3,6,9-methylenedi(n-propyl)phosphate (prepared bythe procedure of Example J). The solution was heated at refl ux for 24 hrs. The reaction mi xture was cooled to room tem perature and extracted with diethyl ether. The ether extract was then concentrated in vacuo to yield the desired productas an off-white solid, 48.6 mg (60%) and characterized by:
0 31p NMR
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).
Toan aqueoussolution of 35mLof lN potassium hydroxidewasadded3.21 9 (3.88mmol)of3,6,9,15-tetraazabicyclo[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 thefiltratefreeze-driedtogiveacreamcoloredsolid. Thesolidwasthensuspensedin 150mL
20 of methanol andstirredfor 12hrsatroomtemperature. Theslurrywasthenfilteredandthe 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 9 (62%) and characterized by:
'H NMR (D2O) 25 ~ 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 13C NMR (D2O) 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, 68.08,1Z5.15, 142.93,152.25; and 30 31p NMR
~9.73 (s, 2P), 21.00 (s, lP).
Example 7 Preparation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca- 1 (15),11,13-tri ene-3[(4-nitrophenyl)methyl acetate]-6,9-methylenediethylphosphonate.
A sol ution of 250 mg (0.62 mmol) of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3[(4-nitrophenyl)methylacetate](preparedbytheprocedureofExampleL), 624 mg (3.7 mmol) of triethyl phosphite, and 111 mg (3.7 mmol) of paraformaldehyde was sti rred at 100C for 1 h r. The reesu Iti ng homogeneous sol uti on was concentrated in vacuo to give a viscous oil. The oil was dissolved in 10 mL of chloroform and washed with water (3 x 5 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered and the filtrate concentrdLed in vacuo to give the product as aviscous oil, 326 mg (96%) and characterized by:
3'P NMR (CDC13) 5 ~ 24.67 (s, 2P), 24.88 (s, 1 P).

BIODISTRIBUTION
General Procedure Sprague Dawley rats were allowed to acclimate for five days then injected with 10 100 llL of the complex solution via a tail vein. The rats weighed between 150 and 200 9 at the time of injection. After 30 min. the rats were killed by cervical dislocation and dissected. The amount of radioactivity in each tissue was determined by counting in a Nal scintillation counter coupled to a multichannel analyzer. The counts were compared to the counts in 100 IlL
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 7/0 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) 20 were evaluated for efficiency of bone localization since phosphonates are known fortheir abilityto bind to hydroxyapatite.
EXAMPLE I
The percent of the injected dose of complex of of Example 2 (1s35m-PCTMP) i n several tissues are given in Table 1. 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 153Sm-PCTMP
TISSUE AVERAGE
Bone 34.87 Liver o gg Kidney 1.42 Spleen 0.07 M uscl e 4.77 Blood 6.27 WO 94/267S4 2 1 6 ~ 1 3 6 PCT/US93/04325 The percent of the injected dose of complex of of Example 5 ('535m-PMPHE) in several tissues are given in Table 11. The numbers represent the average of a minimurn of 3 rats per data point at 2 hours post injection.
TABLE I I
% INJECTED DOSE
535m-PMPH (2 hours) TISSUE AVERAGE
Bone 1 0.86 Liver 4.14 Kidney 1.55 Spleen 0.05 Muscle 1 19 Blood 0.25 15 Heart 0.08 Lung 0.12 Brain 0.00 Stomach 0.44 20 Small Intestine 10.71 Large Intestine 2.17 The percent of the injected dose of complex of of Example 6 ('s3Sm-PMBHE~ in several tissues are given in Table 111. The numbers represent the average of a minimum of 3 rats per data point at 2 hours post injection.

TABLE I I I
% INJ ECTED DOSE
Sm-PMBH . (2 hours) TISSUE AVERAGE
Bone 3.73 Liver 2.70 Kidney 0.43 Spleen 0.05 Muscle 1 .09 0 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 ('s3Sm-PC2Al) in several tissues are given i n Table IV. The numbers represent the average of a mi ni mum of 3 rats per data point at 2 hours post injection TABLE IV
% INJECTED DOSE
53Sm-PC2A 1 P (2 hou rs) TISSUE AVERAGE
Bone 47.98 Liver 1.46 Kidney 0-93 Spleen 0.02 M uscle 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 1 M HCI or NaOH as needed. The total Gd content of each solution was then determined by ICPanalysis.
An anesthetized Sprague Dawley rat was injected i ntramuscularly with one of the metal solutions described above at a dose of 0.05-0.1 mmol Gd/kg body weight. Images werethentakenatvarioustimeintervalsandcomparedwithanon-injectedcontrol attimeO.
Example ll The Gd-PCTMP complex (prepared in Example 2) showed kidney enhancement and bone localization in the shoulder, spine and sternum.
Other embodi ments of the i nvention wil I be apparent to those ski I led i n 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 bythe following claims.

Claims

1. Bicyclopolyazamacrocyclophosphonic acid compounds of the formula (I) wherein:

;

where:
X and Y are independently H, OH, C1-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 more H;
T is H, C1-C18 alkyl, COOH, OH, SO3H, , or :

where: R1 is -OH, C1-C5 alkyl or -O-(C1-C5 alkyl);
R4 is H, NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;
R2 is H or OH; with the proviso that when R2 is OH, then the R term containing the R2 must have all X and Y equal to H;
with the proviso that at least one T must be P(O)R1OH, and with the proviso that when one T is , 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-OR3, C-OR8, N+-R5X-, ;

R3 is H, C1-C5 alkyl, benzyl, or benzyl substituted with at least one R4;
R4 is defined as above;
R4 is C1-C16 alkyl, benzyl, or benzyl substituted with at least one R4;
R8 is C1-C16 alkylamino;
X- is Cl-, Br-, I- or H3CCO2-;
Q and Z independently are CH, N, N+-R5X-, C-CH2-OR3 or C-C(O)-R6-;
R5 is defined as above;
R6 is -O-(C1-C3 alkyl), OH or NHR7;
R7 is C1-C5 alkyl or a biologically active material;
X- is defined as above; or pharmaceutically-acceptable salts thereof;
with the proviso that:
a) when Q, A or Z is N or N+-R5X-, then the other two groups must be CH;
b) when A is C-Br, C-Cl, C-OR3 or C-OR8, then both Q and Z must be CH;
c) the sum of the R4, R7 and R8 terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R6 and when one of Q or Z is C-C(O)-R6, then A
must be CH.
2. A compound of Claim 1 wherein at least two R terms have T equal to P(O)R1OH where R1 is OH and the third T equal H, COOH or C1-C18 alkyl; A, Q and Z are CH; n is 1; and X and Y independently are H or C1-C3 alkyl.
3. A compound of Claim 1 wherein three R terms have T equal to P(O)R1OH
where R1 is OH; and X and Y are H, and named as 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-trimethylenephosphonic acid or pharmaceutically-acceptable salts thereof.
4. A compound of Claim 2 wherein in two R terms T is P(O)R1OH, where R1 is OH, in the third R term T is COOH, and n is 1.
5. A compound of Claim 2 wherein in two R terms T is P(O)R1OH, where R1 is OH, in the third R term T is P(O)R1OH, where R1 is C1-C5 alkyl, and n is 1.
6. A compound of Claim 2 wherein in two R terms T is P(O)R1OH, where R1 is OH, in the third R term T is P(O)R1OH, where R1 is -O-(C1-C5 alkyl), and n is 1.7. A compound of Claim 1 wherein in the R term is at least one T equal P(O)R1OH, where R1 is defined as in Claim 1, and in the other two R terms, T is COOH or P(O)R1OH, and n, R1, X, Y, A, Q and Z are defined as in Claim 1.

8. A compound of Claim 7 wherein in one R term T is P(O)R1OH, where R1 is OH, and in the other two R terms T is P(O)R1OH, where R1 is -O-(C1-C5 alkyl), and n is 1.
9. A compound of Claim 7 wherein in one R term T is P(O)R1OH, where R1 is OH, and in the other two R terms T is P(O)R1OH, where R1 is C1-C5 alkyl, and n is 1.
10. A compound of Claim 7 wherein in one R term T is P(O)R1OH, where R1 is OH, and in the other two R terms T is COOH, and n is 1.
11. A compound of Claim 7 wherein in one R term T is P(O)R1OH where R1 is OH; in the other two R terms T is COOH; n is 1; and X and Y are H; and named as 3,9-diacetic acid-6-(methylenephosphonic acid)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene or pharmaceutically-acceptable salts thereof.
12. A compound of Claim 1 wherein in the R term three T equal P(O)R1OH, where R1 is C1-C5 alkyl or -O-(C1-C5 alkyl), and n, R1, X, Y, A, Q and Z are defined as in Claim 1.
13 A compound of Claim 12 wherein in the three R terms T is P(O)R1OH, where R1 is -O-(C1-C5 alkyl), and n is 1.
14. A compound of Claim 13 wherein in the three R terms T is P(O)R1OH, where R1 is -O-C2H5; and named as 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methyleneethylphosphonate.
15. A compound of Claim 13 wherein in the three R terms T is P(O)R1OH, where R1 is -O-C3H7; and named as 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylene(n-propyl)phosphonate.
16. A compound of Claim 13 wherein in the three R terms T is P(O)R1OH, where R1 is -O-C4H9; and named as 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-methylene(n-butyl)phosphonate 17. A compound of Claim 12 wherein in the three R terms T is P(O)R1OH, where R1 is C1-C5 alkyl, and n is 1.
18. A compound of Claim 1 wherein X and Y are H.
19. A compound of Claim 1 wherein n is 1.
20. A compound of Claim 1 wherein A, Q and Z are CH.
21. A compound of Claim 1 wherein when A, Q and Z are CH, in at least one of the R terms T is other than P(O)R1OH where R1 is OH.
22. A compound of Claim 1 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 or , where R2 and R4 are defined as in Claim 1, and the other two T terms are defined as in Claim 1.
23. A compound of Claim 22 wherein n is 1.

24. A compound of Claim 22 wherein Q, A and Z are CH; and in two R terms X
and Y are H; in one R term X is CO2CH3 and Y is H; n is 1; and one T term is ;

and named as 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3[(4-nitrophenyl)methyl acetate]-6,9-methylenediethylphosphonate.
25. A compound of Claim 22 wherein in 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.
26. A compound of Claim 22 wherein in the two R terms not containing an R4 term, all remaining X and Y terms are H.
27. A compound of Claim 26 wherein in the two R terms not containing an R4 term, both T terms are P(O)R1OH, where R1 is defined as in Claim 1 and is the same moiety.
28. A compound of Claim 26 wherein in the two R terms not containing an R4 term, one T term is a COOH and the other T term is P(O)R1OH, where R1 is defined as in Claim 1.
29 A compound of Claim 1 wherein X and Y are H; T is COOH

or :

where: R1 is -OH, C1-C5 alkyl or -O-(C1-C5 alkyl).
30. A compound of Claim 29 wherein Q and Z are CH.
31. A compound of Claim 30 wherein 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.
32. A compound of Claim 29 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-R6, where R6 is defined as in Claim 1.
33. A compound of Claim 32 wherein R6 is NHR7, where R7 is a biologically active material.
34 A compound of Claim 1 wherein one of A, Q or Z is N+-R5 X-, where R5 and X- are defined as in Claim 1; and in one R term, the T moiety is P(O)R1OH, where R1 is C1-C5 alkyl or -O-(C1-C5 alkyl); and in the other two R terms, the T moiety is P(O)R1OH, where R1 is C1-C5 alkyl, -O-(C1-C5 alkyl) or COOH; and all X and Y terms are H.

35. A compound of Claim 34 wherein in all three R terms, the T moiety is P(O)R1OH, where R1 is C1-C5 alkyl or -O-(C1-C5 alkyl).
36. A complex which comprises a bicyclopolyazamacrocyclophosphonic acid compound as claimed in any one of Claims 1-35;
complexed with a metal ion selected from Gd+3, Mn+2 or Fe+3.
37. A complex of Claim 36 wherein three R terms have T equal to P(O)R1OH
where R1 is OH; and X and Y are H, and named as 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-trimethylenephosphonic acid.
38. A complex as claimed in Claim 36 or 37 wherein the metal is Gd+3.
39. A conjugate comprising a bicyclopolyazamacrocyclophosphonic acid compound as claimed in any one of Claims 1-35, with the proviso that one of R4, R7 or R8 must be present;
complexed with a metal ion selected from Gd+3, Mn+2 or Fe+3; and covalently attached to a biologically active material.
40. A conjugate of Claim 39 wherein the biologically active material is a dextran, a peptide or polypeptide, a molecule that has specific affinity for a receptor, or an antibody or antibody fragment.
41. A conjugate of Claim 40 wherein the antibody or antibody fragment is a monoclonal anti body or fragment thereof.
42. A conjugate of Claim 39 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-R6, where R6 is NHR7, where R7 is a biologically active material.
43. A conjugate of Claim 42 wherein three R terms have T equal to P(O)R1OH
where R1 is OH; and X and Y are H or pharmaceutically-acceptable salts thereof.
44. A conjugate of any one of Claims 30-43 wherein the metal ion is Gd+3.
45. A pharmaceutical formulation comprising a complex of Claim 36 with a pharmaceutically-acceptable carrier.
46. A pharmaceutical formulation comprising a conjugate as claimed in Claim 39 with a pharmaceutically-acceptable carrier.
47. 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 45.48. 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 46. 49. The use of a complex of Claim 36 as a diagnostic agent.
50. The use of a conjugate of Claim 39 as a diagnostic agent.
51. A process for preparing a complex as claimed in Claim 36 which comprises reacting a bicyclopolyazamacrocyclophosphonic acid compound as claimed in Claim 1 with a metal ion selected from Gd+3, Mn+2 or Fe+3 under aqueous conditions at a pH from 5 to 7.

52. The process of Claim 51 wherein the bicyclopolyazamacrocyclophosphonic acid compound is 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-tri-methylenephosphonic acid.
53. 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 hydrogenation or acid.
54. The process of Claim 53 wherein the phosphonating agent has the formula P(OR)3 where R is defined as in Claim 1.
55. The process of Claim 53 wherein the phosphonating agent has the formula P(OR)3 where R is defined as in Claim 1, and formaldehyde in a solvent.
56. Bicyclopolyazamacrocyclophosphonic acid compounds of the formula (I) wherein:

;

where:
X and Y are independently H, OH, C1-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 more H;
T is COOH, or where: R1 is -OH, C1-C5 alkyl or -O-(C1-C5 alkyl);
R4 is H, NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;
R2 is H or OH; with the proviso that when R2 is OH, then the R term containing the R2 must have all X and Y equal to H;
with the proviso that at least one T must be P(O)R1OH where R1 is -O-(C1-C5 alkyl), and with the proviso that when one T is 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-OR3, C-OR8, N+-R5X-, ;

R3 is H, C1-C5 alkyl, benzyl, or benzyl substituted with at least one R4;
R4 is defined as above;
R5 is C1-C16 alkyl, benzyl, or benzyl substituted with at least one R4;
R8 is C1-C6 alkylamino;
X- is Cl-, Br-, I- or H3CCO2;
Q and Z independently are CH, N, N+-R5X-, C-CH2-OR3 or C-C(O)-R6;
R5 is defined as above;
R6 is -O-(C1-C3 alkyl), OH or NHR7;
R7 is C1-C5 alkyl or a biologically active material;
X is defined as above; or pharmaceutically-acceptable salts thereof;
with the proviso that:
a) when Q, A or Z is N or N+-R5X-, then the other two groups must be CH;
b) when A is C-Br, C-Cl, C-OR3 or C-OR8,then both Q and Z must be CH;
c) the sum of the R4, R7 and R8 terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R6 and when one of Q or Z is C-C(O)-R6, then A
must be CH.
57. A compound of Claim 56 wherein all T must be P(O)R1OH where R1 is -O-(C1-C5 alkyl).