CA2139738A1 - Bicyclopolyazamacrocyclocarboxylic acid complexes, their conjugates, processes for their preparation, and use as contrast agents - Google Patents

Abstract

Complexes of bicyclopolyazamacrocyclocarboxylic acid with Gd, Mn or Fe ions are disclosed. The complexes can be covalently attached to a biologically active molecule, e.g. an antibody or antibody fragment, to form conjugates. The complexes and conjugates are useful as contrast agents for diagnostic purposes. Processes for preparing both the complex and conjugate are disclosed.

Description

_ WO 94/2013 213 9 7 3 8 PCTIUS93104322 BICYCLOPOLYAZAMACROCYCLOCARBOXYLIC ACID COMPLEXES, THEIR CONJUGATES, PROCESSES FOR THEIR PREPARATION, AND USE AS CONTRAST AGENTS

This invention concerns complexes that contain as the ligand bicyclopolyaza-5 macrocyclocarboxylic acids, and conjugates thereof, for use as contrast agents i n magnetic :-` i ; resonance imaging (MRI). Processes for preparing these complexes and conjugates are also '. provided. To better understand this invention, a brief background on MRI is provided in the following section.
Backaround , 10 MRI is a non-i nvasive dia~nostic 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 rnathematical equations; see G. M. Barrow, Physical Chemistry,3rd Ed., McGraw-Hill, NY (1973)] to align in an applied magnetic field. Once 15 aligned, this equilibrium state can be perturbed by applying an external radio frequency (RF) pulse which causes the protons to be tilted out of alignment with the magnetic field. When the RF pulse isterminated, 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 - 20 which give information on the degree of molecular organization and interaction of protons with the surrounding environment.
Since water content of living tissue is substantial and variations in content and environment exist among tissue types, diagnostic images of biological organisms are obtained which reflect proton density and relaxation times. The greater the differences in relaxation 25 times (T1 and T2) of protons present in tissue being examined, the greater will be the contrast i n the obtai ned i mage [1. Magnetic Resonance 33,83- 106 (1979)] .
It is known that pa!amagnetic chelates possessing a symmetric electronic ground state can dramatically affectthe T1 and T2 relaxation rates of juxtaposed ~vater protons and that the effectiveness of the chelate in this regard is related, in part, to the number of unpaired 30 electrons producing the magnetic moment [Magnetic Resonance Annual 231 -266 (Raven Press, ~, NY (1985)] . It has also been shown that when a paramagnetic chelate of this type is administereci to a living animal, its effect on lhe T1 and T2 of various tissues can be directly observed in the magnetic resonance (MR) images with increased cont~ast being observed in the areas of chelate localization. It has therefore been proposed that stable, non-toxic 35 paramagnetic chelates be administered to animals in order to increase the diagnostic information obtained by MRI [Frontiers of Biol. Energetics !, 752-759 (1978); J. Nucl. Mecl. 25, 506-513 (1984); Proc. of NMR Imaainq Svmp. (Oct.26-27,1980); F. A. Cotton et al., Adv. Inorg.
i~

~, ;,, '.~

WO 94t26313 ~39~ 3~ PCTIUS93/04322 Chem. 634-639 (1966)]. Paramagnetic metal chelates used in this manner are referred to as contrast enhancement agents or contrast agents.
. There are a nurrlber of paramagnetic metal ions which can be considered when undertaking the design of an MRl contrast agent. In practice, however, the most useful 5 paramagnetic metal ions are gadolinium (Gd ~3), ir~>n (Fe~3), manganese (Mn ~ 2) and (Mn ~ 3), ,-~ and chromium (Cr~3~, because these ions exert the greatest effect on water protons by virtue of `~ their large magnetic moments. In a non-complexed form (e.g. GdC13), these metal ions are toxic to an animal, thereby precluding their use in the simple salt form. Therefore, a fundamental roleoftheorganicchelatingagent(alsoreferredtoasaligand)istorendertheparamagnetic~, 10 metal non-toxic to the animal whi le preserving its desirable influence on T 1 and T2 rel axation rates of the surrounding water protons.
Art in the MRI field is quite extensive, such that the followi ng 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 4,89~,755 discloses a method of alternating the 15 proton NMR relaxation times in the liver or bile duct of an animal using Fe ~3-ethylen~bis(2-hydroxyphenylglycine) complexes and its derivatives, and suggests among various other compounds the possible use of a pyridine macrocyclomethylenecarboxylic acid. U.S. Patent 4,880,008 ~a CIP of U.S. Patent 4,899,755) discloses additional imaging data for livertissue of rats, butwithoutanyadditional complexesbeingshown. U.S. Patent4,980,148disclose 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. Broan et al., J. Chem. Soc., Chem. Commun.,1738-1739 (1990) describe compoundsthataretriazabicyclocompounds. I. K.Adzamli etal.,J. Med. Chem. _~,139-144 (1989) describes acyclic phosphonate derivatives of gadolinium complexes for NMR imaging.
At the present time, the only commercial contrast agent available in the U .S. is the 3 complex of gadolinium with diethylenetriaminepentaacetic acid (DTPA-Gd ' 3 - MAGN EVIST '~
by Sheri ng). MAG N EVIST~ is considered as a non-specificlperf usion agent si nce it freely distributes in extracellular fluid followed by efficient elimination through the renal system.
MAGNEVIST'~ has proven to be extremely valuable in the diagnosis of brain lesions since the " 30 accompanying breakdown of the blood/brain barrier allows perfusion of the contrast agent ' . into the affected regions. In addition to MAGNEVIST'^', Guerbet iscommercially marketing a - macrocyclic perfusion agent (DOTAREM n-) which presently is only available in Europe. A
. number of other potential contrast agents are i n various stages of development.
. It would be advantageous if contrast agents were developed that could have site ' I 35 specificity for the tissue desired to be imaged, rather than non-specific/perfusion agents The i ¦ present invention is directed to just such novel complexes comprising a ligand that is a bicyclo-:, ¦ polya7amacrocyclocarboxylic acid of the formula ,., ~

. r.
.~

21~ 9 ~ ~ 8 PCT/US93/04322 . ~ N
R-N N-R

N
R
`` 10 wherein R is hydrogen, X X

- . -C-CO2H , -C ~ R4 or y CO2H

R
-C ~R4 where: X and Y are independently H, OH, C,-C3 alkyl or COOH;
R7 is H or OH; and Ra is H, NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, 25 bromoacetamido or carboxyl;
with the proviso that at least two R terms must be -C-C02H ;, y A = CH, N, C-Br, C-CI, C-OR', C-OR2, N~-R3 X, or .. -i ~
C-C--C--~ O R4 R' = H, C,-Cs alkyl, benzyl, or benzyl substituted with at least one R~;
R2 j5 C~-CI6 alkylamino;
. .

.~
,, ~", WO 94126313 2~ 39~ 3~ PCT/US93/04322 R3 is Cl-Cl6 alky!, benzyl, or benzyl substituted with at least one R4;
R4 isdefined as before;
X is Cl, Br, 1- or H3CCO2;
Q and Z independently are CH, N, N t-R3 X, C-CH2-ORl or C-C(O)-Rs;
Rl and R3 are defined as above;
Rs is-O-(Cl-C3 alkyl), OH or NHR6;
R6 is C~-Cs al kyl or a biologically active material;
; ~ Xisdefinedasabove;and with the provisos that:
a) when Q, A or Z is N or N -R3 X, then the other two groups must be CH;
b) when A is C-Br, C-CI, C-OR' or C-OR2, then both Q and Z must be CH;
c)thesum of the R2, R4and R6terms, when present, may notexceed one; and d) only one of Q or Z can be C-C~o)-R5 and when one of Q or Z is C-C(O)-Rs, then A
must be CH; and 15 complexed with a metal ion selected from Gd '3, Mn~2 or Fe~3; or pharmaceuticaliy-acceptable salts thereof.
Bifunctional complexes of Formula (I) are desirable to prepare the conjugates ofthis invention. Such ligands must have:
one R term is ~ io , ~ X I R7 R4 or -~- ~R4 CO2H co2 ; where R4 and R' are defined as above; or A is C-OR', C-OR2, where R1 and R2 are defined as above or :;, .
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)-Rs or C-CH2-OR', where R' j and Rs are defined as above;
~ especially those I igands where Rs is NHRs, where R6 is a biologically active material .
The ligands of Formula (I) are complexed with various metal ions, such as gadolinium (Gd ~3), iron (Fe 3), and manganese (Mn'2), and Gd ~3 being preferred. The complexes so formed can be used by themselves or can be attached, by being covalently .";

WO 94/263L3 213 9 7 3 ~ PCT/US93104322 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 conj ugatff of this invention can be modified to provide a :~ 5 specificoverall charge. Forexample,whenthemetal ionis +3thefollowingcanbeobtained:
(A) an overall neutral charge-when i Ris :, X

i, 10 --C--CO2~I
y ~; - and X and Y are all equal to H;
(B) anoverall 1 1charge~when one of A, Q or Z is N ~-R3 X, where R3 and X are defined as above; and the three R
terms are X
~: ~ -C-CO2H
r 20 ~' Y
and X and Y are all equal to H; or when A, Q and Z are CH; X and Y are H; and one R term is H.
-Both the complexes and conjugates may be formulated to be in a I 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 ¦ another copending application.
The complex has the ligand of Formula (I) numbered for nomenclature purposes as follows:

1:',`1 '''' :`~
,~ -5-,~
~ ,~

WO 94/26313 2~9~ 3 P(:TIUS93/04322 14 Q ' ~ z 12 ~ ~s ~ ( I ) ~; R-N 3 6 9 N-R

~ I ~
. R

0 The present invention concerns development of contrast agents having a neutral or + 1 charge which enables site specific delivery of the contrast agent to a desired tissue. The . advantage bei ng i ncreased contrast i n the areas of interest based upon tissue affi nity 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 col1trolled by adjusti ng the total charge and lipophilic character of the complex. The overall range of the charg~ of the complex is from + 1 to 0. For example, for a complex having a + 1 overall charge has heart and/or bone uptake expected; whereas when the overall charge of the complex is 0 (thus neutral), the complex may have the ability to cross the blood brain barrier and normal brain uptake may be possible.
2 Tissue specificity may also be realized by ionic or covalent attachmentof the chelate to a naturally occurring or synthetic macromolecule 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, attachmentof a paramagnetic 2 chelate to a macromolecule can further increase the contrast agent efficiency resulting in imprvved 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 interactic~ns 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 ' ', which may alleviate the discomfort of injection.
While not wishing to be bound by theory, it Is believed that when a charged . complex of the invention is made (e.g. + 1 for heart), the variations in that chelate ionic charge can influence biolocalization. Thus, if the antibody or other directing moiety is also specific for the same site, then the conjugate displays two portions to aid i n site specific delivery.

.

.~., The terms used in Formula (I) and for this invention are further defined as follows.
C1-~3 alkyl , C,-Cs alkyl , C1-C1B alkyl , include both straight and branched chain alkyl groups. An "animal~ includes a warmblooded mammal, preferably a human being.
" Biologically active material N refers to, for example, a dextran, peptide, or f 5 molecules that have specific affinity for a receptor, or preferably antibodies or antibody f ragments.
"Antit~odyn refers to any polyclonal, monoclonal, chimeric antibody or heteroantibody, preferably a monoclonal antibody; "antibody fragment~ incl udes Fab fragments and F(ab')2 fragments, and any portion of an antibody having specificity toward a ~¦ 10 desired epitope or epitopes. When using the term "radioactive metal chelate/antibody ~; j conjugate" or "conjugaten, the "antibodyN is meant to include whole antibodies and/or antibody fragments, including sernisynthetic or genetically engineered variants thereof .
Possible antibodies are 1116-NS-19-9 (anti-colorectal carcinoma), 111~NS-3d (anti-CEA), 703D4 (anti-human lung cancer),704A1 (anti-human lung cancer), CC49 (anti-TAG-72), CC83 (anti-15 TAG-72)andB72.3. Thehybridomacelllinesltl6-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 83û2, ATCC HB 9459, ATCC HB
9453 and ATCC HB 8108, respectively.
As used herein, "cornplexN refers to a complex of the compound of the invention,20 e.g. Formula (i), complexed with a metal ion, where at least one metal atom is chelated or sequestered; "conjugate" refers to a metal ion chelate that is covalently attached to an antibody or antibody fragment. The terms "bifunctional coordinator", ~bifunctional chelating agent" and "functionalized chelant" are used interchangeably and refer to compounds that . have a chelant moiety capable of chelating a metal ion and a moiety covalently bonded to the 25 chelant moiety that is capable of serving as a means to covalently attach to an antibody or antibody fragment.
The bifunctional chelating agentsdescribed 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 30 (represented by R2, R4 or R6 in Formula 1), can be covalently attached to biologically active materials, such as dextran, molecules that have specific affinity for a receptor, or preferably .. ~! coval ently attached to anti bodi es or anti body fragments. Thus the complexes descri bed herei n ;~ ~ may be covalently attached to an antibody or antibody fragment or have specific affinity for a receptor and are referred to herein as "conjugates" .
As used herein, "pharmaceutically-acceptable salt" means any salt or mixtures ofsalts of a complex or conjugate of formula (I) which is sufficiently non-toxic to be useful in J 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 :-s ~ 7 ~i ~ ........... _. .. ..

~,13~l3~3 organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic,succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutami c, gluoonic, _-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic acids and other suitable acids. Also included 5 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 complexes or conjuqates of formula (I) where the salt is potassium, sodium or ammonium. Also included are mi~ures of the above salts.
The complexes or conjugates of the present invention contain a ligand of Formula(I). The ligands are prepared by various processes. Typical general synthetic approaches to such processes are provided bythe reaction schemes given below.
In Scheme 1, the compounds of Formula (I) are prepared wherein Q, Aand Z =
CH, and either one R = H and the other two R = the formula below or all three R =
X

: :
' ~. ::
~. ~

,, ~
- ~ ~ 25 ~: .

. ~

' ;'~''1 . .i,":' WO 94/26313 PCT/USg3/04322 U~
E~

< Z Z ~ _ ., ~ ~ X
Z~
E~ ~,\Q' h Z U~ C~
ræ--E~ o Lz--E~ ~: ~ æ z--m Z ~ ~ Z

E <~ = I O

u U m z X X
U~ o J ~ ~
~ ~ -- ~1 ~ ~ m C~ ~ Z -- 5: o o t~ /~ >=~o X ~ \ Z Z ~
m \~ ~ o <~ o ~ z _/ e~
< z ~ m ,5 S ~ m l' ,~

~7 _9_ WO 94/26313 ~ 39~ 3~

~ ~, i~ . ' ) ~ Z _ , ~ ~ U~
z~ / m v ~ m ~; o ~ Z-~

E C~
~ m 3 U ~ ~ o C~ :~
~ o z~ E~
~' ~/ >

~ X

J ~ -lO-WO 94/26313 2 I 3 9 7 3 8 PCTIUS~3/04322 -Scheme 2 prepares the compounds of Formula (I) wherein A = C-Br, and Q
and Z - CH.

~f7 'I ' ."~
,.1 ' .. ..

~,~33~13~
Z) t o ~ o ~ .

i, .
~ / _ ~, m~\ Z co m~z ~ ô

I O ~
V
.1~ S ~1 .
o V
11 ~

LZ--~ 5 m o ~ _ _ r m~=\z ~

),L o ~ ~
Y~ ~ ~

":, -.~

-:C

50~ o o o ::C
~ .
N U O
m t) N
O
3 V ~ ~

m ~ Z~ o I; r I ~
~ ,..~1 WO 94/26313 13~ PCTIUS93/04322 Scheme 3 prepares the compounds of Formula (I) wherein A =

C-C--C~R4 R" = H, NOz, NH2 or SCN; and Q and Z = CH.

;, .

~ .,, ,, -14-213973~
;

.... ...

1. :, .;.. .~

,, ~
,: ~
: ~ ~ Ul E~
- ~ I

: ~ ~ ~, . ~ ~
t~ H I~
P. ~ ~

~,' ~i ~ ~5 -15-r ~

WO 94/26313 ~ 3S

o .
C~
o ~ :Z~ ~) H

o E

V C~
~, V~ o~ o N ~ æ

. - 1 ,. -16-'~1 WO 94126313 213 9 7 ~ PCTIUS93104322 J Scheme 4 prepares the compounds of Formula (I) wherein A - C-OR2, where R2 = Cl-C5 alkylamino; and QandZ = CH.
,~ .

:, .~

~ ~ PCTIUS93/04322 WO 94/26313 ?. ~39~ ~ ~

~' ~ Z

o o ~ ~
Z=c T~ ô
~,- ' ~ C O~æ ~

,, ~ rz ~ ~ u ' : ~ ~ Z ' ~ ~

~ 0~
~0 = ~ ~

~r ~: j æ--, ,,, WO 94/263L~ 213 9 7 3 8 PCT/US931û4322 ,. ~, $
:~. o~
-s Z~

~ ZV -C~ .
O

. O
N ~
::C
'. ~ 5: ~ o~

~ ~Z-) E O ~z 1 ~ ~ m 1~ . o Z~ ~ rz~

o~\ Z ~--~

Z
I' ~ .
! ' ~ x ¦ ,' m ~

;! -19-WQ 94/26313 ~ 3 ;, 3 ~

U

O
~r U~ o :.., :, .s, ': _ '' ..

. .

., ' . .

'r ~, !

-~ 21~9738 Scheme 5 prepares the com pounds of Formula (I) wherein A _ CH; and r~ one of Q or Z = CH and the other Q or Z = C C(O)-R6 or C-CH2-R6, where R6 is defi ned as before.
i WO 94126313 2~3 PCT/US93/04322 rzz~0 ~0 æ \ m x U7 \ ~ ~

O

su ~Z ~ \
~ t ~ ~ 1 Z o ~ O E~
I

U~
U E~

, - .
.
~...."~
.~ ....

, . ~. ........ . . . . . .. . . . . . . .

¦ . 21?9738 '., t ' ~, ~ .

. ' ~ O
~ ~'1 O :~:
: C~
:: ~ m~ o Y U Z
o m " m E
. 3- ~

~a WO 94/26313 ~3 9~ PCT/US93/04322 . S{heme 6 prepares the compounds of Formula (I) wherein Z = C-CH2-OBz or C-C(O)-Rswhere Rs = --(Cl-C3 alkyl), OH or NHRs, where is defined as before; and Q and ~ = CH

. .

: .

~, .

<

o --m o~
O ~ ~
p~
~ EC ~ ~

~ E~ O ~ ~

L~ ' I
..

n PCT/US93/04322 WO 94/26313 ~ 3 9~ 3 ~3 O < _ ~

j (~ )$ E,~
\/u O

o u o $ ~ ~ ~

~17 S H

< ~7 ~,J O

O
_~ ~

o7 o 0~
' :,' U ~ ~C
~Iml ?

~ .- X Z

!
:

Scheme 7 prepares the compounds of Formula ~1) wherein A = N or N ~-Rs -~i X; Rs = C~-C16 alkyl and is X ha!ide; and QandZ = CH.

., ;;,.
;:;
:~ --27--3 9 7 ~j~

i~ o ~ 1 ~z-~ ~
~ I ~ o X
r ~t ~ ~
~,. =z-~ ~ .
u~ ~
--Z--E~ ~:
a Z

~: ~ mz_ V~ Z-~
C,~ :C

~"', ;~ ix . oo ~
~C.
. ~
Z_ : :
r7 . C~
~J ~
i Z Z ~
. I ~, ., ~, ....

213973x m ~ H
:' O
.,., ~ ~tl5 - ;~ X ~r ) -,i ~--Z Z ~ ~ O

O

m ~ ~O
m~ ~1 ~e m I ~ o t :1:
1~ ,~
. ~ /
. .~ ~ ~
r Z; Z ~0 Z_ O

o ~ o o X
x a~ x i o . ... I X
_~ Z
~r ; ~

:~ -29-~,~ 39~l3~

Scheme 8 prepares the compounds of Formula (I) wherein Q = N~-Rs X, where Rs = Cl-Ct6 alkyl and X = halide; and A and Z = CH.

. ;

.

,~
, :
.", ; ~
:~

,~

'~
~ ' :

:
:
:

1-, .~

WO 94/26313 21~ 9 7 3 ~ PCT/US93/04322 .

_ ; -~ ~ rZ-ptl ~,v~z ~
~ Z~
, a~ z C~ E~

o r~ O X

c ~ j X ~ ~ m C~
~ ~ ~Z ~ , rl l :~
.i .
:~ -31-'.
, :~

WO 94/26313 2 ~ 9~ 3 ~ PCTIUS93/04322 " .

~ H
I ~ _ ~
\z -- O

X ~ O

i: ~ ` O

a~ ~ ~

N L 1~
C.) 11 O
XX Z

--:~ -32-2:1~97~8 I
~, `- Scheme 9 prepares the compounds of Formula (I) wherein Q = N or N + -Rs X-, where Rs = C1-cl6 alkyl and i X = halide; and A and Z = CH.

.' 1 .

.~ , .

/

' J i ~1~
, ~ -33-~3g~3~

::C H
O

<

<~ U ~ ~

m ~C ~
~ r r~ ~ ~ ~
O
~ / O
~ ~D \ In ~ U r Z~
E x c X ~ / ..

O _.

< -- -- O
r ~ ~ m ~Z
;~- o < ~:
~ o 3~ C~
o ~ ~
o o U 1 2;
:r m ~
o I x ~a ,1 ` .

., .~. - 3 4 -~ ~. . . . . . . .

WO 94/26313 213 9 7 3 ~ PCT/US93/04322 . Scheme 10 prepares the compounds of Forrnula (I) wherei n R terrn at the 6 position Is X
-C--(~ R4 where R4 = NO2 or NH2; and A,QandZ = CH.

!

:,~

~ WO 94/26313 PCT/US93/04322 2~39~3~
'-;
., ' '~
,. _ O
~z o~
r Z--, Z
~Z ~ ~ + ,~r o~ ~
Z
_ Z_ U
~ ~ U~
~Z ~ + ~Z~

m ~ .. ~ .

~ ~ -36-r i~

:3 .~ .

~

.. ~ o t o e ,. ~ ~ c~
~ . ~
~ ~, o o, ~ I ~ o~

~ > O
. ~ $
~ I
o o '\
~ m ~

C~ o xXx In I ~ _ WO 94/26313 . PCTtUS93/04322 ~39~3~

Scheme 11 prepares the compounds of Formula (I) wherein the R term at the 9 position is where R4 = NO2 or NH2; and A,QandZ = CH.

WO 94/26313 21 3 9 7 3 ~ PCT/US93/04322 ,-- z ~
O
~z j~ -o ~ m c~

o 1: ~ o I

o~",~ "

~ Z
o o r \ ~ ~
C) m m <~ ) O O

: I o ~, l ., ",;. .~

WO ~4/26313 ~,~313~ 3~ PCTIUS93/043~2 Scheme 12 prepares the compounds of Formula (I) wherein n = 1 (but would also apply if n = 2 or 3 with the corresponding change in the reagent), the R
. . . term at the 6 position has T =
.---P--0~;
-where R1 _ {)H; and X and Y = H;
the R term atthe 3 and 9 positions have T = COOH; and A,QandZ = CH.

.

, .

.. .- . , ::~;

~ -40-WO 94/26313 21 3 9 73 ~ PCT/US93/04322 -'i .

:.~

:i :! z .. o ~ r~
,=~, z ~

<~z z~

~ z o \z ~ ~ t ~ r z ~ _m ~

O Z--L
z +

Z--r~ ~ _ <\ Z Z
Z _>
!

. 1 .. .
~. -.. ,, -. - . - - . . .

2~39~
' , ~, ~, Z~
,, Z

r ~ O
~ Z>~o Lz ~
_ A ~ ~ ~ Ç--l ~ m ~

a~
" o - o I ~

~ ;,,t~
L
C~
o ,, o :~

,. ...

In the above Schemes, the general process description illustrates specific stepsthat 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 astreatment with toluenesuifonyl chloride, HBr or HCI, 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. Or~. Chem. 54, 299~2992 (1989)]. Detosylation of the intermediate macrocycle 1(3) to yield (4)1 was accomplished under acidic conditions in good yield. Reductive detosylation procedures are also wel I known i n the literature and can be adapted to the present reaction sequence.
Schemes 10, 11 and 12 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 carborlate. Additional f unctionalization of the remaining nitrogen positions is then preformed by methods and conditions described in previous Schemes. After the introduction of the desired chelating moieties, the nitro group is reduced using platinum oxide and hydrogen in water. Inthis form, thechelating agentiscompatiblewith conjucJationtechniqueswhichwill enable attachment to larger synthetic or natural molecules.
The metal ions used to form the complexes of this invention are Gd ~ 3, Mn ~ 2, Fe 3 and available comrnercially, 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 spinangular momentum and/or orbital angular momentum. The two types of momentum combine togive the observed paramagnetic moment in a mannerthat depends largely on the atoms bearing the unpaired electron and, to a lesser extent, upon the environment of such atoms The paramagnetic nuclides found to be useful in the practice of the invention are gadolinium (Gd~3), iron (Fe~3) and manganese (Mn~2), with Gd~3 being preferred~
The complexes are prepared by methods well known in the art~ Thus, for exarnple, see Chelating Agents and Metal Chelates, Dwyer & Mellor, Academic Press (1964), Chapter 7. See also methods for making amino acids in Svnthetic 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 ,~.,,' wo g4a63~ 9 ' 3 PC~N593/043~2 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 composition, e.g. stableto 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 iigand 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 conjuqates ~idescribed herein can be prepared by techniques well known in the art. Highly specific '10 monoclonal antibodies can be produced by hybridization techniques well known in the art, see -for example, Kohler and Milstein INature, 256,495-497 t 1975); and Eur. J. Immunol., 6, 511 -519 (1976)1. Suchantibodiesnormallyhaveahighlyspecificreactivity. Intheantibodytargeted 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 15 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. 50me examples of antibodies or antibody fragments are 1116-NS- 19-9,1 t l 6-NS-3d, 703D4, 704A1, CC49, CC83 20 and B72.3. All of these antibodies have been deposited in ATCC. A more complete list of antigens can be found in U.S. Patent 4,193,983. The conjugates of the present invention are particularly preferred forthe diagnosis of various cancers.
This invention is used with a physiologically acceptable carrier, excipient or -vehicle therefor. The methods for preparing such formulations are well known. The 25 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 fordiagnosis. The dose will vary depending on the disease and physical parameters of the animal, such as weight. In vivo 30 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, attachmentto polymeric supports for various purposes, e.g. as diagnostic agents, and removal of metal ion by selective extraction.
The ligands of Formula (I) having in at least two R terms T equal to P(O)R'OH may be used for 35 metal ion control as scale inhibitors. It is likely that these ligands could be used in less than stoichiometric amounts. Similar uses are known for compounds described in U.S. Patents 2,609,390; 3,331,773; 3,336,221; and 3,434,969.

,~ -44-'~1 ,, : ,, , The invention wi 11 be further clarified by a consideration of the following exampies, which are intended to be purely exemplary of the present invention.
Some terms used in the following examples are defined as follows:
t LC = liquid chromatrography, purifications were carrier out at low pressure using Dionex 2010i system fitted with a hand-packed Q-Sepharose'~ anion exchange column (23 x 2 cm).
DMF = dimethylformamide.
AcOH = aceticacid.
ICP = inductively coupled plasma.
9 = gram(s).
mg~ milligrams.
. kg = kilogram~s).
mL = milliliter(s).
}IL = microliter(s).

pH Stabilitv General Procedure A stock 's9GdC13 or 153SmCI3 solution was prepared by addinci 2 ~lL of 3x10~M
9GdCI3 in 0.1 N HCI to 2 mL of a 3x1 04M GdC13 carrier solution. Appropriate ligand solutions were then prepared in deionized water. The 1:1 ligand/metal complexes were then prepared - 20 by combining the ligands (dissolved in 100-500 yL of deionized water) with 2 mL of the stock ~A ~59GdCI3 solution, followed by through mixing to give an acidic solution (pH = 2). The pH of the '',J solution was then raised to 7.0 using 0.1 N NaOH. The percent metal as a complex was then determined by passing a sample of the complex solution through a Sephadex'~ G-50 column, eluting with 4:1 saline (85% NaCI/NH~OH) and collecting 2 x 3 mL fractions. The amount of 25 radioactivity in the combined elutions was then compared with that left on the resin (non-i, complexed metal is retained on the resin). The pH stability profilewas generated by adjusting ~ the pH of an aliquet of the complex solution using 1 M NaOH or 1 M 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 ex,c ermintal comparison to be identical for complexation and 30 biodistribution of the ligands of this invention.
~I STARTING MATERIALS
t 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) 35 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 i, temperature, the solid which formed was filtered, washed with benzene and dried in vacuo.
~1 ';5 -45-~,..

WO 94/26313 213 9 ~ 38 PCT/US93/04322 The solid was then neutralized with saturated NaHCO3, filtered and dried to yietd 23.1 g (71.5%) of the titled product as an off-white crystalline solid, rnp74.5-75.5C, and further characterized by:
'H NMR (CDCI3) ~ 1 5 ~ 4.88 (s,4H),7.25-7.95 (m,3H).
I ~ ExamPle B
Preparationof 3,6,9-tris(~tolylsulfonyl)-3,6,9,15-tetraazabicyclo~9.3.1]pentadeca-1(15),11,13-` ~ ; triene.
A DMF solution (92 mL) of 6.9 9 (11.4 mmol) of 1,4,7-tris(~
o tolylsulfonyl)diethylenetriamine disodium 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 bythe procedure of Example A) in 37 mL of DMF. When I i ~ 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 NaCi, followed 15 by precipitation of the title product. The resuiting 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- 170C dec. and further characterized by:
~, -: ' H NMR (CDCI3) ~ 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 i 20 (d, 2H),7.65 (d,4H),7.75 (t,1 H); and ~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
25 Preparationof3,6,9,15-tetraazabicyclo~9.3.1]pentadeca-1(15),11,13-triene.
A solution of HBr and AcOH was prepared by mixing 48% HBr and ~lacial AcOH in ; a 64: 35 ratio. To 112 mL of the HBr/AcOH mixture was added 5.5 9 (8.2 mmol) of 3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclol9.3.1]pentadeca-1(15),11,13-triene(preparedbythe procedure of Example B) and the reaction mixture was heated at mild reflux with constant 30 stirring for 72 hrs. The reaction mixture was then cooled to room temperature and concentrated to approximately 1/1û of the original voliJme. 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 tetrahydrc bromide salt was then dissolved in 10 mL of water, adjusted to pH 9.5 with NaOH (50% w/w) and continuously 35 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 j roorn temperature to yield 1.2 g (71 %) of the title product, mp 86-88C and further - characterized by:
:~ -46-:~

WO 94/26313 213 9 7 s~ 8 PCT/US93/04322 - 'H 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, lH); and t 13C NMR
48.73,49.01, 53.63,119.67,136.29,159.54.
. 5 Example D
Preparationof3,6,9,15-tetraazabicyclo[9.3.11pentadeca-1(15),11,13-triene-3,6,9-triaceticacid ~ (PCTA).
- An aqueous solution ~15 mL) of 2.1 9 (15 mmol) of bromoacetic acid was added to 0.8 9 (3.8 mmol) of 3,6,9,15-tetraazabicyclol9.3.1 lpentadeca-1 (15),11,13-triene (prepared by ~ 10 theprocedureofExampleC)withstirringatroomtemperature. Aftercompletedissolution, .~ the reaction mixture was cooled with an ice bath and the pH adjusted to 9 by the slow additic n of NaOH (50%wlw). The pH was held constant at 9 throughout the reaction by addi ng small aliquotsof NaOH. After 1.5hrsthereaction mixturewaswarmedto60Cwithcontinued monitoring of pH. When no further drop in pH could be detected, the reaction was cooled to 15 room temperature and the aqueous solution freeze-dried to give a white solid . The solid was then dissolved i n a minimum of hot water and allowed to stand at room temperature f or 12 - hrs. The resulting crystalswere filtered and dried in vacuotogive 1.2 9 (70%) of the title product as the trisodium salt, mp 378-380C dec. and further characterized by:
:
'H NMR (D2O) 20 ~ 2.76 (m,4H), 3.36 (m,4H),3.47 (s,2H),4.10 (s,4H),7.31 (d,2H),7.84 (t,1 H); and S3.83, 57.31, 57.40,59.48,62.36,125.47,143.72,152.67,172.15,177.41.
ExamDle E
Preparation of 3,9-bis(sodium methylenesulfonate)-3,6,9,15-tetraazabicyclo[9.3.1 Ipentadeca-25 1 (15),11,13-triene (PC2S).
A solution of 10 mL of an aqueous solution of 1.03 9 (5~0 mmol) of 3,6,9,15-tetraazabi cyclol9.3.1 lpentadeca- 1 (15),11,13-tri ene (prepared by the proced u re of Exampl e C) and 0.5 mL of concentrated HCI was stirred for 10 min at room temperature. The solution had a pH of 8.6. To the solution was then added 1.37 9 (10.2 mmol) of HOCH2SO3Na and 5 m L of 30 deionized water. The solution was then heated at 60~C for 10 min and the pH was 5.6. After cooling, the pH was adjusted to 9.0 with 1 M NaOH, followed by freeze-drying to give the desired product as a white solid (quantitative yield), and further characterized by:
, i 'H NMR (D70) 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 35 '3C NMR (D2O) ~48.52, 54.04,58.92,75.09,123.90,141.37,161.89.
~' :
~. -47-WO 94126313 2 ~-~ 9~ 3~ PCT/US93/04322 Examnle F
Preparation of 3,9-bis(methylenenitrile)-3,6,9,1~tetraazabicyclol9.3.1~pentadeca-1(15),11,13-triene.
` To an aqueous solution of 10 mL of 3,g-bis(sodium methylenesulfonate)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(prepared bytheprocedureof Example E) was added 10 mL of ~.06 g (12.24 mmol) of NaCN. The reaction mixture was stirred for 3 hrs at room temperature. The solution had a pH of about 10. Upon adjustment of the pH to greater than 13 by with concentrated NaOH, the product precipitated, was extracted with chloroform (3 x 20 mL~, dried over magnesium sulfate, and filtered. Upon removal of the solvent and ' 10 concentrationinvacuo,thedesiredproductwasisolatedasawaxywhitepowder,1.û0g(71%), and further charanerized by:
H NM R (CDCI 3) 2.03 (s br,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) ~46.64,52.89,60.78,115.31,122.02,137.57,157.33.
Exam~le G
Preparation of 3,6,9,15-tetraazabicyclot9.3.1]pentadeca-1(1S),11,13-triene-3,9-dimethylenenitrile-6-(2-methoxy-5-nitrophenyl)methyl acetate.
To 7 mL of a THF solution of 200 mg (0.73 mmol) of 3,6,9,15-20 tetraazabicyclo[9;3.1 ~pentadeca- 1 (15),11,13-triene-3,9-dimethylenenitri le ~prepared by the procedure of Example F) was added 223 mg (0.73 mmol) of bromo-(2-methoxy-5-nitrophenyl)methyl acetate. The resulting solution was stirred at room temperature for 12 hrs.
To the reaction mixture was added 100 mg of K2CO3 and the mixture stirred for an additional 2 hrs. The reaction mixture was then filtered and the filtrate concentrated in vacuo. The 25 resulting crude product was ~hen purified by column chromatography (silica gel, 5%
CH30H/CHCI3).
Example H
Preparation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,9-aceticacid-6-(2-methoxy-5-nitrophenyl)acetic acid.
3,6,9,15-Tetraazabicyclol9.3.1~pentadeca-1(15),11,13-triene-3,9-dimethylenenitrile-6-(2-methoxy-5-nitrophenyl)methyl acetate (prepared by the procedure of - ExampleG)wasstirredforl2hrsatrefluxin6NHCI. Thesolutionwasthencooledand concentrated in vacuo. The residue was then dissolved in water and Iyophilized to give the desired product.
Example I
,5 Preparation of 3,9-diacetic acid-3,6,9,15-tetraazabicyclol9.3.1]pentadeca-1(15),11,13-triene , (PC2A) ,: .
., ~

WO 94/26313 213 9 7 3 g PCT/IJS93104322 A concentrated aqueoussolution of 30 mL of HCI (37~) and mg (2.5 mmol) of 3,9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclol9.3.1]pentadeca-1(15),11,13-triene(prepared . by the procedure of ExampJe F) was heated at reflux for 2 hrs. After cooling, the aqueous solution was evaporated to dryness, followed by coevaporation with deionized water (2 x 10 5 mL) to eliminate excess HCI. The pH of the reaction mixture was adjusted to 7 with -1 concentrated NaOH. The resulting nutral solution chromatographed on cation exchange ~SP-? Sepharose'~) column ~1.5 x 50 cm), elutin with first deionized water, then with 1 M HCI. The acidic fraction containing product was evaporated to dryness, followed by coevaporation with ,; deionized water (3 x 10 mL) to el i mi nate excess HCI . The fi nal product was isolated as a white '- t0 solid upon freeze drying of the concentrated aqueous solution, and characterized by:
'H NMR (D2O) 2.84 (s br, 4H),3.18 (m, 4H), 3.77 (s, 4Hj,4.35 (s, 4H),7.63 (d, 2H),8.23 (t,1 H); and '3C NMR (D2C)) ~47.45, 54.33, 59.73, 60.36,127.20, 149.31, 155.60,177.74.

ExamDle 1 Preparati on of the complex of l53Sm-3,6,9,15-tetraazabicycl o[9.3.1]pentadeca- 1 t 15),11,13-triene-3,6,9-trimethylenecarboxylic acid ('s3Sm-PCTA) A solution of the ligand of Example D was prepared by dissolving 3.8 mg of 20 ligand/0.517mLofdeionizedwater(pH=2). A1:1 ligand/metalcomplexwasthen prepared by combining40~lLoftheligandsolutionwith2mLofaqueousSmCI3H20(3xlO4MinO.01N HCI) containing tracer l53SmCI3. 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/NH~OH), and collecting 2 x 3 mL fractions. The amount of 25 radioactivity in the combi ned elutions was then compared with that left on the resi n. U nder 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 ~2%. A sample of the sol ution that was passed through the resin was used for pH studies. The pH stabil ity was then determined using the General Procedure above.
Complexation for the title product after passing through the resin was determinedtobegreaterthan98% atthe 1:1 ligandtometal ratio.
BIODISTRIBUTION
General Procedure Sprague Dawley rats were allowed to acclimate for five days then injected with 100 yL of the complex solution via a tail vein. The rats weighed between 150 and 200 g at the time of injection. After 30 min. the rats were killed by cervical dislocation and dissected. The amount of radioactivity in each tissue was determined by counting in a Nal scintillation counter ~ -49-~ .;

2l39~3~

coupled to a multichannel analyzer. The counts were compared to the counts i n 100 ~lL
standards i n order to determine the percentage of the dose in each tissue or organ.
The percent dose in biood was estimated assuming blood to be 7% of the body weight. The percent dose in bone was esti mated by muitipuling the percent dose i n the fem ur 5 by 25. The percent dose in mus~le was estimated assuming muscle to be 43% of the body ~n ~ ~ - weight.
- i In addition .to organ biodistribution, chelates of the compounds of Formula (I) -' were evaluated for efficiency of bone localization since phosphonates are known fortheir ` ~ ability to bind to hydroxyapatite.
o EXAMPLE I
The percent of the injehed dose of complex of of Example 1 (ls3Sm-PCTA) in several tissues are given in Table 1. The numbers representthe average of ~ rats per data point.
TABLE I
`~ ~; % INJECTED DOSE IN SEVERAL
TISSUES FOR Sm-PCTA
: 15Tissue Average :
:: . Bone 2.77 Liver 0.80 Kidney 1.50 0Spleen 0.12 .
Muscle 0.87 Blood 0.39 . ~ The bone to blood ratio (/O dose) was 7. The bone to liver ratio was 3.5. The bone ; ~ ~: to muscle ratio was 4.8.
~ 25 r''~ IMAGING EXPERIMENTS
General Procedure injectable so!utions were first prepared (0.SM) by dissolving the appropriate amount of each complex in 2 mL of deionized water. The pH of the solutions were then 30 adjusted to 7.4 using 1 M HCI or NaOH as needed. The total Gd content of each soiution 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-i njected control at time 0.
Ex~amDle ll . ~. The Gd-PCTA complex (prepared in Example 1~ was rapidly taken up by the renal : system with brilliant enhancement of the kidney cortex as well as peripheral kidney tissue.
"

:~.
; ~
!, Other embodiments of the inven~ion will be apparent to those skiIled 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.

., ; -51 -,~

Claims

WHAT IS CLAIMED IS:

1. A complex which comprises a bicyclopolyazamacrocyclocarboxylic acid compound of the formula (I) wherein:
R is hydrogen, , or ;

where:
X and Y are independently H, OH, C1-C3 alkyl or COOH;
R7 is H or OH; and R4 is H, NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;
with the proviso that at least two R terms must be ;

A = CH, N, C-Br, C-Cl, C-OR1, C-OR2, N+-R3 X-, or ;

where: R1 = H, C1-C5 alkyl, benzyl, or benzyl substituted with at least one R4;
R2 is C1-C16 alkylamino;
R3 is C1-C16 alkyl, benzyl, or benzyl substituted with at least one R4;
R4 is defined as before;
X- is Cl-, Br-, I- or H3CCO2-;
Q and Z independently are CH, N, N+-R3X-, C-CH2-OR1 or C-C(O)-R5;
R1 and R3 are defined as above;
R5 is -O-(C1-C3 alkyl), OH or NHR6;
R6 is C1-C5 alkyl or a biologically active material;
X- is defined as above; and with the proviso that:
a) when Q, A or Z is N or N+-R3X-, then the other two groups must be CH;
b) when A is C-Br, C-Cl, C-OR1 or C-OR2, then both Q and Z must be CH;
c) the sum of the R2, R4 and R6 terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R5 and when one of Q or Z is C-C(O)R5, then A
must be CH; and complexed with a metal ion selected from Gd+3, Mn+2 or Fe+3; or pharmaceutically-acceptable salts thereof.
2. A complex of Claim 1 wherein the metal is Gd+3.
3. A complex of Claim 1 wherein A, Q and Z are CH; and X and Y are H.
4. A complex of Claim 1 wherein X and Y are H.
5. A complex of Claim 1 wherein A, Q and Z are CH.
6. A complex of Claim 1 wherein Q, A and Z are CH; and one R term is or where: X, Y, R2 and R4 are defined as in Claim 1.
7. A complex of Claim 1 wherein A is C-OR1, C-OR2, where R1 and R2 are defined as in Claim 1 or where R4 is defined as in Claim 1.
8. A complex of Claim 1 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-R5 or C-CH2-OR1, where R1 and R5 are defined as in Claim 1.
9. A complex of Claim 8 wherein R5 is NHR6, where R6 is a biologically active material.
10. A conjugate comprising a bicyclopolyazamacrocyclocarboxylic acid compound of the formula (I) wherein:
R is hydrogen, , or ;

where:
X and Y are independently H, OH, C1-C3 alkyl or COOH;
R7 is H or OH; and R4 is H, NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;
with the proviso that at least two R terms have a CO2H group present A = CH, N, C-Br, C-Cl, C-OR1, C-OR2, N+-R3X-, or ;

R1 = H, C1-C5 alkyl, benzyl, or benzyl substituted with at least one R4;
R2 is C1-C16 alkylamino;
R3 is C1-C16 alkyl, benzyl, or benzyl substituted with at least one R4;
R4 is defined as before;
X- is Cl-, Br-, I- or H3CCO2-;
Q and Z independently are CH, N, N+-R3X-, C-CH2-OR1 or C-C(O)-R5;
R1 and R3 are defined as above:
R5 is -O-(C1-C3 alkyl), OH or NHR6;
R6 is C1-C5 alkyl or a biologically active material;
X is defined as above; and with the proviso that:
a) when Q, A or Z is N or N+-R3X-, then the other two groups must be CH;
b) when A is C-Br, C-Cl, C-OR1 or C-OR2, then both Q and Z must be CH;
c) the sum of the R2, R4 and R6 terms, when present, may not exceed one; and d) only one of Q or Z can be C-C(O)-R5 and when one of Q or Z is C-C(O)-R5, then A
must be CH;
complexed with a metal ion selected from Gd+3, Mn+2 or Fe+3; and covalently attached to a biologically active material.
11. A conjugate of Claim 10 wherein the biologically active material is a dextran, a peptide, a molecule that has specific affinity for a receptor, or an antibody or antibody fragment.
12. A conjugate of Claim 11 wherein the antibody or antibody fragment is a monoclonal antibody or fragment thereof.
13. A conjugate of Claim 12 wherein the antibody or antibody fragment is B72.3.
14. A conjugate as claimed in any one of Claims 10-13 wherein the metal ion is Gd+3.
15. A conjugate of Claim 10 wherein X and Y are H
16. A conjugate of Claim 10 wherein A, Q and Z are CH.
17. A conjugate of Claim 10 wherein Q, A and Z are CH; and one R term is where: X and R4 are defined as in Claim 10.
18. A conjugate of Claim 10 wherein Q, A and Z are CH; and one R term is where: R4 and R7 are defined as in Claim 10.
19. A conjugate of Claim 10 wherein A is C-OR1, C-OR2, where R1 and R2 are defined as in Claim 10, or ;

where R4 is defined as in Claim 10.
20. A conjugate of Claim 10 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-R6, where R6 is defined as in Claim 10.
21. A conjugate of Claim 20 wherein R6 is NHR7, where R7 is a biologically active material.
22. A pharmaceutical formulation comprising a complex as claimed in any one of Claims 1-9 with a pharmaceutically-acceptable carrier.
23. A pharmaceutical formulation comprising a conjugate as claimed in any one of Claims 10-21 with a pharmaceutically-acceptable carrier.
24. A method for the diagnosis of a disease state in an animal which comprises administering to said animal an effective amount of the formulation of Claim 22.25. A method for the diagnosis of a disease state in an animal which comprises administering to said animal an effective amount of the formulation of Claim 23.26. The complex as claimed in any one of Claims 1-9 for use as a pharmaceutical.
27. The conjugate as claimed in any one of Claims 10-21 for use as a pharmaceutical.
28. A kit for use as a diagnostic agent having as an ingredient a ligand as claimed in any one of Claims 1-9.

29. A process for preparing a complex as claimed in Claim 1 which comprises reacting a bicyclopolyazamacrocyclocarboxylic 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.
30. The process of Claim 29 wherein the bicyclopolyazamacrocyclocarboxylic acid compound is 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-tri-methylenecarboxylic acid.