Note: Descriptions are shown in the official language in which they were submitted.
_ 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
,., ~
-2-
. r.
.~
21~ 9 ~ ~ 8 PCT/US93/04322
WO 94126313
. ~ N
R-N N-R
N
R
`` 10 wherein
R is hydrogen,
X X
- . -C-CO2H , -C ~ R4 or
y CO2H
R
-C ~R4
CO2H
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;
. .
- 3 -
.~
,, ~",
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
-4-
.";
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
1 35
'''' :`~
,~ -5-
,~
~ ,~
WO 94/26313 2~9~ 3 P(:TIUS93/04322
14 Q ' ~ z 12
~ ~s ~ ( I )
~; R-N 3 6 9 N-R
14 N
~ 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.
-6-
.
.~.,
WO 94/26313 213 9 7 3 8 PCT/US93/04322
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
~ ........... _. .. ..
WO 94/26313 PCT/US93/04322
~,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
C-CO2H
: :
' ~. ::
~. ~
,, ~
- ~ ~ 25
~: .
. ~
' ;'~''1
8-
. .i,":'
2139738
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_
PCTIUS93/04322
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 '
.. ..
WO 94/26313 PCT/US93/04322
~,~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~ ~ ~
":,
12-
WO 94/26313 PCT/US93/04322
2139738
-.~
-
:C
50~ o
o o ::C
~ .
N U O
m
t)
N
O
3 V ~ ~
m ~ Z~ o
I; r
I ~
~ ,..~1
-13-
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-
WO 94/26313 PCT/US93/04322
213973~
;
.... ...
1. :,
.;.. .~
,, ~
,: ~
: ~ ~ Ul
E~
- ~ I
: ~ ~ ~,
. ~ ~
t~ H I~
P. ~ ~
~,'
~i
~ ~5 -15-
r ~
PCT/US93/04322
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.
,~ .
:,
.~
-17-
~ ~ 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-
PCT/IJS93/04322
WQ 94/26313 ~ 3 ;,
3 ~
U
O
~r
U~ o
:..,
:,
.s,
': _
''
..
. .
.,
' . .
'r
-20-
~, !
WO 94/26313 PCT/US93/04322
-~ 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
-21-
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
WO 94/26313 PCT/US93/04322
'.,
t
' ~,
~ .
. ' ~ O
~ ~'1
O :~:
: C~
:: ~ m~ o
Y U Z
o m
" m
E
. 3- ~
~a
-23-
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
. .
: .
-24-
~,
WO 94/26313 213 9 7 3 8 PCT/US93/04322
.
<
o
--m
o~
O ~ ~
p~
~ EC ~ ~
~ E~ O ~ ~
L~ '
I
..
-25-
n PCT/US93/04322
WO 94/26313 ~ 3 9~ 3 ~3
7 0
O < _ ~
j (~ )$ E,~
\/u O
o
u o
$ ~ ~ ~
~17
S H
< ~7
~,J O
O
_~ ~
o7 o 0~
' :,' U ~ ~C
~Iml ?
~ .- X Z
!
:
-26-
WO 94/25313 213 9 7 3 8 PCT/US93/04322
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--
WO 94126313 PCT/US93/04322
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 ~,
.,
~, ....
-28-
WO 94/26313 PCT/US93/04322
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-
WO 94126313 PCTIUS93/04322
~,~ 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-,
-30-
.~
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-
WO 94126313 PCT/US93/04322
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-
WO 94/26313 PCT/US93/04322
~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
C0
where R4 = NO2 or NH2; and
A,QandZ = CH.
!
:,~
-35-
~ 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-
WO 94/26313 PCT/US93/04322
2139738
r
i~
:3
.~ .
~
.. ~ o
t o e
,. ~ ~ c~
~ . ~
~ ~,
o o,
~ I ~ o~
~ > O
. ~ $
~ I
o o '\
~ m ~
C~ o
xXx
In
I ~ _
-37-
WO 94/26313 . PCTtUS93/04322
~39~3~
Scheme 11 prepares the compounds of Formula (I) wherein
the R term at the 9 position is
R4
C0
where R4 = NO2 or NH2; and
A,QandZ = CH.
-38-
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
., ",;. .~
--39--
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
-41 -
.. .
~. -.. ,, -. - . - - . . .
PCT/IJS93/04322
WO 94/26313
2~39~
'
, ~,
~, Z~
,, Z
r ~ O
~ Z>~o
Lz ~
_ A ~ ~ ~ Ç--l
~ m ~
a~
" o - o
I ~
~ ;,,t~
L
C~
o
,, o :~
,. ...
42-
WO 94/26313 21 3 9 7 3 8 PCT/US93/04322
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
-43-
,~.,,'
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
,, : ,,
WO 94/26313 2 1 3 9 7 3 8 PCTIUS93104322
,
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
3C NMR
~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
3C NMR
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)
-48-
,: .
., ~
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.
15 FINAL PRODUCTS
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~
WO 94/263L3 PCT/US93/04322
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.
"
50-
:~.
; ~
!,
WO 94/26313 213 91 3 8 PCT/U593/04322
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 -
,~
