U.S. patent application number 10/741872 was filed with the patent office on 2004-07-08 for enhanced relaxivity monomeric and multimeric compounds.
Invention is credited to Pillai, Radhakrishna, Ranganathan, Ramachandran S., Ratsep, Peter C., Shukla, Rajesh, Tweedle, Michael F., Zhang, Xun.
Application Number | 20040131551 10/741872 |
Document ID | / |
Family ID | 22909910 |
Filed Date | 2004-07-08 |
United States Patent
Application |
20040131551 |
Kind Code |
A1 |
Ranganathan, Ramachandran S. ;
et al. |
July 8, 2004 |
Enhanced relaxivity monomeric and multimeric compounds
Abstract
Metal chelates capable of exhibiting an immobilized relativity
between about 60 and 200 mM.sup.-1s.sup.-1/metal atom are useful as
magnetic resonance imaging agents. Additionally, a compound which
is useful as a metal-chelating ligand has the following formula: 1
wherein Q is a 4- to an 8-membered carbocyclic ring which may be
fully or partially saturated; t is an integer from 2 to 16; each R
group is independently hydrogen, --OH, --CH.sub.2-A,
--OCH.sub.2CH(OH)CH.sub.2-A or a functional group capable of
forming a conjugate with a biomolecule, provided that at least two
of the R groups are selected from --CH.sub.2-A or
--OCH.sub.2CH(OH)CH.sub.2-A; and A is a moiety capable of chelating
a metal atom.
Inventors: |
Ranganathan, Ramachandran S.;
(Princeton, NJ) ; Pillai, Radhakrishna; (Kendall
Park, NJ) ; Ratsep, Peter C.; (Hamilton Square,
NJ) ; Shukla, Rajesh; (Lawrenceville, NJ) ;
Tweedle, Michael F.; (Princeton, NJ) ; Zhang,
Xun; (Kendall Park, NJ) |
Correspondence
Address: |
KRAMER LEVIN NAFTALIS & FRANKEL LLP
INTELLECTUAL PROPERTY DEPARTMENT
919 THIRD AVENUE
NEW YORK
NY
10022
US
|
Family ID: |
22909910 |
Appl. No.: |
10/741872 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10741872 |
Dec 19, 2003 |
|
|
|
08241253 |
May 11, 1994 |
|
|
|
6693190 |
|
|
|
|
Current U.S.
Class: |
424/9.363 ;
534/16; 540/465; 540/474 |
Current CPC
Class: |
C07D 493/10 20130101;
C07D 257/02 20130101; C07D 405/14 20130101; C07F 9/6524 20130101;
A61K 49/06 20130101 |
Class at
Publication: |
424/009.363 ;
534/016; 540/465; 540/474 |
International
Class: |
A61K 049/00; C07F
005/00 |
Claims
What is claimed is:
1. A compound of the following formula I: 28wherein each m, n, o
and p is independently 1 or 2; q is 0 or 1; each G is independently
--COOR", --P(O)(OR").sub.2, --P(O)(OR")(R") or --C(O)N(R").sub.2;
each R' is independently hydrogen or alkyl, alkoxy, cycloalkyl,
hydroxyalkyl or aryl, each of which is optionally substituted, or a
functional group capable of forming a conjugate with a biomolecule
or of forming a multimer of said compound of formula I; each R" is
hydrogen; each R.sup.13 through R.sup.20 is independently hydrogen,
alkyl, hydroxyalkyl, alkoxyalkyl or a functional group capable of
forming a conjugate with a biomolecule or of forming a multimer of
said compound of formula I; or R.sup.13 together with R.sup.15, and
R.sup.17 together with R.sup.18 , independently form, together with
the carbon atoms in the tetraazacyclododecane macrocycle to which
they are attached, a fused fully or partially saturated
non-aromatic cyclohexyl ring which may be unsubstituted or
substituted by one or more halogen, alkyl, ether, hydroxy or
hydroxyalkyl groups, and which may be further fused to a
carbocyclic ring, or R.sup.13 and R.sup.15, are each hydrogen and
R.sup.17, together with R.sup.18, forms a fused fully or partially
saturated non-aromatic cyclohexyl ring as defined above, or
R.sup.13, together with R.sup.15, forms a fused fully or partially
saturated non-aromatic cyclohexyl ring as defined above and
R.sup.17 and R.sup.18 are hydrogen; provided that (a.) when G is
always --COOR" and (i.) R', R", R.sup.14 and R.sup.16 through
R.sup.20 are all hydrogen, then R.sup.13 and R.sup.15 are other
than hydrogen; (ii.) R" and R.sup.13 through R.sup.20 are all
hydrogen, and m, n, o, p and q are each 1, then (CR'R') is other
than (CH.sub.2) and (CHCH.sub.3); (iii.) R', R", R.sup.13,
R.sup.14, R.sup.17 and R.sup.20 are all hydrogen, then at least two
of R.sup.15, R.sup.16, R.sup.18 and R.sup.19 are other than methyl;
and (iv.) R", R.sup.16, R.sup.19 and R.sup.20 are all hydrogen, and
each (CR'R') is independently (CHR') or (CH.sub.2CHR'), then
R.sup.13 and R.sup.15, and R.sup.17 and R.sup.18, are other than a
fused ring; and (b.) when G is always --P(O)(OR").sub.2,
--P(O)(OR")(R" ) or --C(O)N(R").sub.2, then at least one R' or
R.sup.13 through R.sup.20 is other than hydrogen; or a salt or
multimeric form thereof.
2. A metal chelate, comprising a compound of claim 1 complexed with
a metal atom.
3. The chelate of claim 2, wherein the metal is selected from atoms
having an atomic number of 21 to 29, 42 or 57 to 83.
4. The chelate of claim 3, wherein said metal is gadolinium.
5. A compound of the following formula II: 29wherein Q is a 4- to
an 8-membered carbocyclic ring which may be fully or partially
saturated; t is an integer from 2 to 16; each R group is
independently hydrogen, --OH, --CH.sub.2-A,
--OCH.sub.2CH(OH)CH.sub.2-A or a functional group capable of
forming a conjugate with a biomolecule, provided that at least two
of the R groups are selected from --CH.sub.2-A or
--OCH.sub.2CH(OH)CH.sub.2-- A; and A is a moiety capable of
chelating a metal atom.
6. A compound of claim 5 wherein A is 30each m, n, o and p is
independently 1 or 2; q is 0 or 1; each G is independently --COOR",
--P(O)(OR").sub.2, --P(O)(OR")(R") or --C(O)N(R").sub.2; each R' is
independently hydrogen or alkyl, alkoxy, cycloalkyl, hydroxyalkyl
or aryl, each of which is optionally substituted, or a functional
group capable of forming a conjugate with a biomolecule or of
forming a multimer; each R" is hydrogen; each R.sup.13 through
R.sup.20 is independently hydrogen, alkyl, hydroxyalkyl,
alkoxyalkyl or a functional group capable of forming a conjugate
with a biomolecule or of forming a multimer of said compound of the
formula I; or R.sup.13 together with R.sup.15, and R.sup.17
together with R.sup.18, independently form, together with the
carbon atoms in the tetraazacyclododecane macrocycle to which they
are attached, a fused fully or partially saturated non-aromatic
cyclohexyl ring which may be unsubstituted or substituted by one or
more halogen, alkyl, ether, hydroxy or hydroxyalkyl groups, and
which may be further fused to a carbocyclic ring, or R.sup.13 and
R.sup.15 are each hydrogen and R.sup.17, together with R.sup.18,
forms a fused fully or partially saturated non-aromatic cyclohexyl
ring as defined above, or R.sup.13, together with R.sup.15, forms a
fused fully or partially saturated non-aromatic cyclohexyl ring as
defined above and R.sup.17 and R.sup.18 are hydrogen; provided that
(a.) when G is always --COOR" and (i.) R', R", R.sup.14 and
R.sup.16 through R.sup.20 are all hydrogen, then R.sup.13 and
R.sup.15 are other than hydrogen; (ii.) R" and R.sup.13 through
R.sup.20 are all hydrogen, and m, n, o, p and q are each 1, then
(CR'R') is other than (CH.sub.2) and (CHCH.sub.3); (iii.) R', R",
R.sup.13, R.sup.14, R.sup.17 and R.sup.20 are all hydrogen, then at
least two of R.sup.15, R.sup.16, R.sup.18 and R.sup.19 are other
than methyl; and (iv.) R", R.sup.16, R.sup.19 and R.sup.20 are all
hydrogen, and each (CR'R') is independently (CHR') or
(CH.sub.2CHR'), then R.sup.13 and R.sup.15, and R.sup.17 and
R.sup.18, are other than a fused ring; and (b.) when G is always
--P(O)(OR").sub.2, --P(O)(OR")(R") or --C(O)N(R").sub.2, then at
least one R' or R.sup.13 through R.sup.20 is other than hydrogen;
or a salt or multimeric form thereof.
7. A compound of claim 5, wherein A is 31each R' is independently
hydrogen, alkyl, alkoxy, hydroxyalkyl, aryl, aralkyl or arylalkoxy;
each R" is hydrogen; and each n is 1 or 2.
8. A compound of claim 5 wherein 32is 33each R.sup.1 through
R.sup.12 group is independently hydrogen, --OH, --CH.sub.2-A,
--OCH.sub.2CH(OH)CH.sub.2-A or a functional group capable of
forming a conjugate with a biomolecule; at least two of R.sup.1
through R.sup.12 are selected from --CH.sub.2-A or
--OCH.sub.2CH(OH)CH.sub.2-A; and R.sup.8 and R.sup.9 taken together
may additionally form the group --O--[C(RR)]--O-- where each R is
independently hydrogen or alkyl, or R.sup.8 and R.sup.9 taken
together may form 34
9. A compound of claim 6 wherein 35is a compound of the formula
36wherein each R.sup.1 through R.sup.12 group is independently
hydrogen, --OH, --CH.sub.2-A, --OCH.sub.2CH(OH)CH.sub.2-A or a
functional group capable of forming a conjugate with a biomolecule;
at least two of R.sup.1 through R.sup.12 are selected from
--CH.sub.2-A or --OCH.sub.2CH(OH)CH.sub.2-A; and R.sup.8 and
R.sup.9 taken together may additionally form the group
--O--[C(RR)]--O-- where each R is independently hydrogen or alkyl,
or R.sup.8 and R.sup.9 taken together may form 37
10. A compound of claim 7, wherein 38is 39each R.sup.1 through
R.sup.12 group is independently hydrogen, --OH, --CH.sub.2-A,
--OCH.sub.2CH(OH)CH.sub.2-A or a functional group capable of
forming a conjugate with a biomolecule; at least two of R.sup.1
through R.sup.12 are selected from --CH.sub.2-A or
--OCH.sub.2CH(OH)CH.sub.2-A; and R.sup.8 and R.sup.9 taken together
may additionally form the group --O--[C(RR)]--O-- where each R is
independently hydrogen or alkyl, or R.sup.8 and R.sup.9 taken
together may form 40
11. A metal chelate, comprising a compound of claim 5 complexed
with a metal atom.
12. A metal chelate, comprising a compound of claim 6 complexed
with a metal atom.
13. A conjugate, comprising a compound of claim 5 conjugated with a
biomolecule.
14. A metal chelate, comprising a conjugate of claim 13 complexed
with a metal atom.
15. A method for diagnostic imaging, comprising the steps of
administering to a host a compound of claim 5, which compound is
complexed with a metal, and obtaining a diagnostic image of said
host.
16. The method of claim 15, wherein said image is a magnetic
resonance image.
17. A method for diagnostic imaging, comprising the steps of
administering to a host a conjugate of claim 13, which conjugate is
complexed with a metal, and obtaining a diagnostic image of said
host.
18. A compound selected from the group consisting of:
(1.alpha.,2.alpha.,4.beta.,5.beta.)-10,10'-[(1,2,4,5-Tetrahydroxy-1,4-cyc-
lohexanediyl)bis(methylene)]bis[1,4,7,10-tetraazacyclododecane-1,4,7-triac-
etic acid];
(3a.alpha.,4.alpha.,5.beta.,6.alpha.,7.beta.,7a.alpha.)-10,10'-
10",10"'-[[Hexahydro-2,2-dimethyl-1,3-benzodioxol-4,5,6,7-tetrayl]tetra(ox-
y)-tetra(2-hydroxy-3,1-propanediyl)]tetrakis[1,4,7,10-tetraazacyclododecan-
e-1,4,7-triacetic acid];
3,4,5,6-Tetra-O-[2-hydroxy-3-[4,7,10-tris(carboxy-
methyl)-1,4,7,10-tetraazacyclododecan-1-yl]propyl]-myo-inositol;
(1.alpha.,2.alpha.,3.alpha.,4.beta.,5.alpha.,6.beta.)-10,10'-[(2,3,5,6-te-
trahydroxy-1,4-cyclohexanediyl)bis(2-hydroxy-3,1-propanediyl)]-bis[1,4,7-1-
0-tetraazacyclododecane-1,4,7-triacetic acid]; 10
[[1,4-Dihydroxy-2,5-bis[-
2-hydroxy-3-[4,7,10-tris-(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-y-
l]-propoxy]-4-[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-
-yl]methyl]cyclohexyl]methyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacet-
ic acid;
[3aR-(3a.alpha.,3"a.alpha.,4.beta.,4".beta.,5.alpha.,5".alpha.,6.-
beta.,6"b,7.alpha.,-7".alpha.,7a.alpha.,7"a.alpha.)]-dodecahydro-4,4",5,5"-
,6,6",7,7"-octakis-[[2-hydroxy-3-[(4,7,10-tricarboxymethyl)-1,4,7,10-tetra-
azacyclododecan-1-yl]propyl]-oxy]dispiro-[1,3-benzodioxole-2,1'-cyclo-hexa-
ne-4',2"-[1,3]-benzodioxole];
[2S-(2.alpha.,5.alpha.,8.alpha.,11.alpha.)]--
2,5,8,11-Tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid;
[aR-(.alpha.R*,.alpha.'R*,.alpha."R*,.alpha.'"R*,2S*,5S*,8S*,11S*)]-
-.alpha.,-.alpha.',.alpha.",a'",2,5,8,11-Octamethyl-1,4,7,10-tetraazacyclo-
dodecane-1,4,7,10-tetraacetic acid;
[2S-(2R*,5R*,8R*,11R*)]-2,5,8,11,-Tetr-
amethyl-1,4,7,10-tetraazacylcododecane-1,4,7-triacetic acid; and
10-(Phosphonomethyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic
acid.
19. A compound selected from the group consisting of:
(1.alpha.,2.alpha.,4.beta.,5.beta.)-10,10'-[(1,2,4,5-Tetrahydroxy-1,4-cyc-
lohexanediyl)bis(methylene)]bis[1,4,7,10-tetraazacyclododecane-1,4,7-triac-
etic acid], digadolinium salt;
(3a.alpha.,4.alpha.,5.beta.,6.alpha.,7.beta-
.,7a.alpha.)-10,10'10",10"'-[[Hexahydro-2,2-dimethyl-1,3-benzodioxol-4,5,6-
,7-tetrayl]tetra(oxy)-tetra(2-hydroxy-3,1-propanediyl)]tetrakis[1,4,7,10-t-
etraazacyclododecane-1,4,7-triacetic acid], tetragadolinium salt;
3,4,5,6-Tetra-O-[2-hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaz-
acyclododecan-1-yl]propyl]-myo-inositol, tetragadolinium salt;
(1.alpha.,2.alpha.,3.alpha.,4.beta.,5.alpha.,6.beta.)-10,10'-[(2,3,5,6-te-
trahydroxy-1,4-cyclohexanediyl)bis(2-hydroxy-3,1-propanediyl)]-bis[1,4,7-1-
0-tetraazacyclododecane-1,4,7-triacetic acid], digadolinium salt;
10-[[1,4-Dihydroxy-2,5-bis[2-hydroxy-3-[4,7,10-tris-(carboxymethyl)-1,4,7-
,10-tetraazacyclododecan-1-yl]-propoxy]-4-[[4,7,10-tris(carboxymethyl)-1,4-
,7,10-tetraazacyclododecan-1-yl]methyl]cyclohexyl]methyl]-1,4,7,10-tetraaz-
acyclododecane-1,4,7-triacetic acid, tetragadolinium salt;
[3aR-(3a.alpha.,3"a.alpha.,4.beta.,4".beta.,5.alpha.,5".alpha.,6.beta.,6"-
b,7.alpha.,-7".alpha.,7a.alpha.,7"a.alpha.)]-dodecahydro-4,4",5,5",6,6",7,-
7"-octakis-[[2-hydroxy-3-[(4,7,10-tricarboxymethyl)-1,4,7,10-tetraazacyclo-
dodecan-1-yl]propyl]-oxy]dispiro-[1,3-benzodioxole-2,1'-cyclo-hexane-4',2"-
-[1,3]-benzodioxole], octagadolinium salt; [2S-
(2.alpha.,5.alpha.,8.alpha-
.,11.alpha.,)]-2,5,8,11-Tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,1-
0-tetraacetic acid, gadolinium salt;
[.alpha.R-(aR*,.alpha.'R*,.alpha."R*,-
a'"R*,2S*,5S*,8S*,11S*)]-.alpha.,
-.alpha.',.alpha.",a'",2,5,8,11-Octameth-
yl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid,
gadolinium salt;
[2S-(2R*,5R*,8R*,11R*)]-2,5,8,11,-Tetramethyl-1,4,7,10-tetraazacylc-
ododecane-1,4,7-triacetic acid, gadolinium salt; and
10-(Phosphonomethyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7-triacetic
acid, gadolinium salt.
20. A chelate other than
[1R-(1R*,4R*,7R*,10R*)]-.alpha.,.alpha.',.alpha."-
,.alpha.'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid or
[1R-(1R*,4R*,7R*)]-.alpha.,.alpha.',.alpha."-trimethyl-1,4,7
,10-tetraazacyclododecane-1,4,7-triacetic acid possessing a
stability greater than 10.sup.15 M.sup.-1 and capable of exhibiting
an immobilized relaxivity between about 60 and 200
mM.sup.-1s.sup.-1/metal atom.
21. A chelate of claim 20 capable of exhibiting an immobilized
relaxivity between about 70 and 150 mM.sup.-1s.sup.-1/metal
atom.
22. A chelate of claim 20 capable of exhibiting an immobilized
relaxivity between about 80 and 100 mM.sup.-1s.sup.-1/metal
atom.
23. A chelate possessing a stability greater than 10.sup.15
M.sup.-1 and capable of exhibiting an immobilized relaxivity
between about 60 and 200 mM.sup.-1s.sup.-1/metal atom comprising a
compound of claim 1.
24. A chelate possessing a stability greater than 10.sup.15
M.sup.-1 and capable of exhibiting an immobilized relaxivity
between about 70 and 150 mM.sup.-1s.sup.-1/metal atom comprising a
compound of claim 1.
25. A chelate possessing a stability greater than 10.sup.15
M.sup.-1 and capable of exhibiting an immobilized relaxivity
between about 80 and 100 mM.sup.-1s.sup.-1/metal atom comprising a
compound of claim 1.
26. A chelate possessing a stability greater than 10.sup.15
M.sup.-1 and capable of exhibiting an immobilized relaxivity
between about 60 and 200 mM.sup.-1s.sup.-1/metal atom comprising a
compound of claim 8.
27. A chelate possessing a stability greater than 10.sup.15
M.sup.-1 and capable of exhibiting an immobilized relaxivity
between about 70 and 150 mM.sup.-1s.sup.-1/metal atom comprising a
compound of claim 8.
28. A chelate possessing a stability greater than 10.sup.15
M.sup.-1 and capable of exhibiting an immobilized relaxivity
between about 80 and 100 mM.sup.-1s.sup.-1/metal atom comprising a
compound of claim 8.
29. A chelate other than
[1R-(1R*,4R*,7R*,10R*)]-.alpha.,.alpha.',.alpha."-
,.alpha.'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid or [1R*,4R*,7R
*)]-.alpha.,.alpha.',.alpha."-trimethyl-1,4,7,10-tetr-
aazcyclododecane-1,4,7-triacetic acid, including an immobilized
functional group, capable of exhibiting an immobilized relaxivity
between about 60 and 200 mM.sup.-1s.sup.-1/metal atom.
30. A chelate of claim 29, capable of exhibiting an immobilized
relaxivity between about 70 and 150 mM.sup.-1s.sup.-1/metal
atom.
31. A chelate of claim 29, capable of exhibiting an immobilized
relaxivity between about 80 and 100 mM.sup.-1s.sup.-1/metal
atom.
32. A chelate including a functional group, capable of exhibiting
an immobilized relaxivity between about 60 and 200
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 1.
33. A chelate including a functional group, capable of exhibiting
an immobilized relaxivity between about 70 and 150
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 1.
34. A chelate including a functional group, capable of exhibiting
an immobilized relaxivity between about 80 and 100
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 1.
35. A chelate including a functional group, capable of exhibiting
an immobilized relaxivity between about 60 and 200
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 8.
36. A chelate including a functional group, capable of exhibiting
an immobilized relaxivity between about 70 and 150
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 8.
37. A chelate including a functional group, capable of exhibiting
an immobilized relaxivity between about 80 and 100
mM.sup.1s.sup.-1/metal atom comprising a compound of claim 8.
38. A chelate other than
[1R-(1R*,4R*,7R*,10R*)]-.alpha.,.alpha.',.alpha."-
,.alpha.'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid, conjugated to a biomolecule, capable of exhibiting an
immobilized relaxivity between about 60 and 200
mM.sup.-1s.sup.-1/metal atom.
39. A chelate of claim 38, capable of exhibiting an immobilized
relaxivity between about 70 and 150 mM.sup.-1s.sup.-1/metal
atom.
40. A chelate of claim 38, capable of exhibiting an immobilized
relaxivity between about 80 and 100 mM.sup.-1s.sup.-1/metal
atom.
41. A chelate conjugated to a biomolecule, capable of exhibiting an
immobilized relaxivity between about 60 and 200
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 1.
42. A chelate conjugated to a biomolecule, capable of exhibiting an
immobilized relaxivity between about 70 and 150
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 1.
43. A chelate conjugated to a biomolecule, capable of exhibiting an
immobilized relaxivity between about 80 and 100
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 1.
44. A chelate conjugated to a biomolecule, capable of exhibiting an
immobilized relaxivity between about 60 and 200
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 8.
45. A chelate conjugated to a biomolecule, capable of exhibiting an
immobilized relativity between about 70 and 150
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 8.
46. A chelate conjugated to a biomolecule, capable of exhibiting an
immobilized relaxivity between about 80 and 100
mM.sup.-1s.sup.-1/metal atom comprising a compound of claim 8.
Description
BRIEF DESCRIPTION OF THE INVENTION
[0001] In accordance with the present invention, novel monomeric
and multimeric compounds having enhanced relaxivities are provided.
These compounds are useful, for example, as metal-chelating
ligands. The compounds are also useful in the form of metal
complexes as diagnostic contrast agents. When the metal in the
complex is paramagnetic, the diagnostic contrast agents are
especially suitable for magnetic resonance imaging (MRI).
[0002] In one embodiment of the invention, certain specific
compounds comprise a tetraazacyclododecane macrocycle, and are
represented by the formula I: I 2
[0003] wherein
[0004] each m, n, o and p is independently 1 or 2;
[0005] q is 0 or 1;
[0006] each G is independently --COOR", --P(o)(OR").sub.2, --P(O)
(OR") (R") or --C(O)N(R").sub.2;
[0007] each R' is independently hydrogen or alkyl, alkoxy,
cycloalkyl, hydroxyalkyl or aryl, each of which is optionally
substituted, or a functional group capable of forming a conjugate
with a biomolecule or of forming a multimer of said compound of
formula I;
[0008] each R" is hydrogen;
[0009] each R.sup.13 through R.sup.20 is independently hydrogen,
alkyl, hydroxyalkyl, alkoxyalkyl or a functional group capable of
forming a conjugate with a biomolecule or of forming a multimer of
said compound of the formula I;
[0010] or R.sup.13 together with R.sup.15, and R.sup.17 together
with R.sup.18, independently form, together with the carbon atoms
in the tetraazacyclododecane macrocycle to which they are attached,
a fused fully or partially saturated non-aromatic cyclohexyl ring
which may be unsubstituted or substituted by one or more halogen,
alkyl, ether, hydroxy or hydroxyalkyl groups, and which may be
further fused to a carbocyclic ring, or R.sup.13 and R.sup.15 are
each hydrogen and R.sup.17, together with R.sup.18, forms a fused
fully or partially saturated non-aromatic cyclohexyl ring as
defined above, or R.sup.13, together with R.sup.15, forms a fused
fully or partially saturated non-aromatic cyclohexyl ring as
defined above and R.sup.17 and R.sup.18 are hydrogen; provided that
(a.) when G is always --COOR" and (i.) R', R", R.sup.14 and
R.sup.16 through R.sup.20 are all hydrogen, then R.sup.13 and
R.sup.15 are other than hydrogen; (ii.) R" and R.sup.13 through
R.sup.20 are all hydrogen, and m, n, o, p and q are each 1, then
(CR'R') is other than (CH.sub.2) and (CHCH.sub.3); (iii.) R', R",
R.sup.13, R.sup.14, R.sup.17 and R.sup.20 are all hydrogen, then at
least two of R.sup.15, R.sup.16, R.sup.18 and R.sup.19 are other
than methyl; and (iv.) R", R.sup.16, R.sup.19 and R.sup.20 are all
hydrogen, and each (CR'R') is independently (CHR') or
(CH.sub.2CHR'), then R.sup.13 and R.sup.15, and R.sup.17 and
R.sup.18, are other than a fused ring; and (b.) when G is always
--P (O)(OR").sub.2, --P(O)(OR")(R") or --C(O)N(R").sub.2, then at
least one R' or R.sup.13 through R.sup.20 is other than
hydrogen;
[0011] or a salt or multimeric form thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0012] Listed below are definitions of various terms used in the
description of this invention. These definitions apply to the terms
as they are used throughout this specification, unless otherwise
limited in specific instances, either individually or as part of a
larger group.
[0013] The expression "relaxivity" refers to the effectiveness of a
metal chelate to reduce the relaxation time of bulk water in
contact with the metal chelate.
[0014] The expression "immobilized relaxivity" refers to the
relaxivity measured when a chelate moiety can undergo only slow
molecular reorientation because of rigid attachment to a large
moiety or a physiological surface, or because it is dissolved in a
medium of high viscosity.
[0015] The expression "water relaxivity" refers to relaxivity in
water where a chelate moiety possesses a relaxivity dominated by
overall molecular reorientation.
[0016] The expression "enhanced relaxivity" refers to relaxivity
values made greater than those of well characterized prior art
molecules by 1.) altering the electronic relaxation rate,
.tau..sub.s, through modifications of the metal-donor atom bond
vibration frequencies and/or amplitudes, (this being accomplished,
for example, by increasing the steric bulk and/or orientation of
organic elements bonded to the donor atoms and/or the macrocyclic
carbon atoms), 2.) in a multimer by decreasing the internal
molecular motion of one monomer unit relative to another (this
being accomplished, for example, by increasing the steric bulk of
the organic groups linking the monomer units) or 3.) by decreasing
the molecular reorientation of a monomer or a multimer attached to
a large moiety or a physiological surface.
[0017] The term "stability" refers to the equilibrium formation
constant (K) of the reaction M+L.fwdarw.M(L) where
K=[M(L))]/[M][L], M is a metal ion, L is a chelating ligand and
M(L) is a chelate complex of a metal and a ligand.
[0018] The term "alkyl" refers to both straight and branched,
unsubstituted chains of carbon atoms. Those chains having 1 to 5
carbon atoms are preferred. Methyl is the most preferred alkyl
group.
[0019] The term "cycloalkyl" refers to cyclic hydrocarbon groups of
3 to 8 carbon atoms. The groups may be unsubstituted or substituted
by, for example, alkyl, halogen, hydroxy, hydroxyalkyl, alkoxy,
alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino,
alkanoylamino, thiol, alkylthiol, nitro, cyano, carboxy, carbamoyl,
alkoxycarbonyl, alkylsulfonyl, sulfonamido and the like.
[0020] The term "alkoxy" refers to -alkyl(O). Methoxy is the most
preferred alkoxy group.
[0021] The term "aryl" refers to phenyl, pyridyl, furanyl,
thiophenyl, pyrrolyl, imidazolyl and the like, all of which may be
substituted. Preferred substituted aryl groups are those
substituted with 1, 2 or 3 halogen, nitroamino, maleimido,
isothiocyanato, hydroxy, hydroxyalkyl, alkyl, alkoxy, carbamoyl,
carboxamide, acylamino or carboxy moieties.
[0022] "Hydroxyalkyl" refers to straight and branched alkyl groups
including one or more hydroxy radicals such as
--CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OHCH.sub.2OH, --CH
(CH.sub.2OH).sub.2 and the like. (See, for example, Sovak, M.,
Editor, Radiocontrast Agents, Springer-Verlag, 1984, pp.
1-125).
[0023] The term "aralkyl" refers to an aryl group bonded through an
alkyl group.
[0024] The term "carbocyclic ring" refers to a ring system in which
all the ring atoms are carbon, e.g., phenyl or cyclohexyl. The ring
may be unsubstituted or substituted by, for example, alkyl,
halogen, hydroxy, hydroxyalkyl, alkoxy, alkanoyl, alkanoyloxy,
amino, alkylamino, dialkylamino, alkanoylamino, thiol, alkylthiol,
nitro, cyano, carboxy, carbamoyl, alkoxycarbonyl, alkylsulfonyl,
sulfonamido and the like.
[0025] The term "halogen" refers to bromo, chloro, fluoro or
iodo.
[0026] The term "alkanoyl" refers to the group alkyl-C(O)--.
[0027] The term "alkanoyloxy" refers to the group
alkyl-C(O)--O--.
[0028] The term "amino" refers to the group --NH.sub.2.
[0029] The term "alkylamino" refers to the group --NHR where R is
alkyl.
[0030] The term "dialkylamino" refers to the group --NRR' where R
and R' are each, independently, alkyl.
[0031] The term "alkanoylamino" refers to the group
alkyl-C(O)--NH--.
[0032] The term "thiol" refers to the group --SH.
[0033] The term "alkylthiol" refers to the group --SR where R is
alkyl.
[0034] The term "nitro" refers to the group --NO.sub.2.
[0035] The term "cyano" refers to the group --CN.
[0036] The term "carboxy" refers to the group --C(O)OH or the group
--C(O)OR where R is alkyl.
[0037] The term "alkoxycarbonyl" refers to the group
alkoxy--C--(O)--.
[0038] The term "alkylsulfonyl" refers to the group
alkyl-SO.sub.2.
[0039] The term "sulfonamido" refers to the group
--SO.sub.2NH.sub.2, the group --SO.sub.2NHR or the group
--SO.sub.2NRR' where R and R' are each, independently, alkyl,
cycloalkyl or aryl.
[0040] The term "carbamoyl" refers to the group --C(O)NH.sub.2, the
group --C(O)NHR or the group --C(O)NRR' where R and R' are each,
independently, alkyl, alkoxy or hydroxyalkyl.
[0041] The term "carboxamide" refers to the group --C(O)NH.sub.2,
the group --C(O)NHR or the group --C(O)NRR' where R and R' are
each, independently, alkyl.
[0042] The term "acylamino" refers to the group --NH--C(O)--R where
R is alkyl.
[0043] The expressions "bioactive group" and "bioactive moiety"
denote a group which is capable of functioning as a metabolic
substrate, catalyst or inhibitor, or is capable of being
preferentially taken up at a selected site of a subject, such as by
possessing an affinity for a cellular recognition site.
[0044] When compounds of the formula I are in the multimeric form,
each monomer is preferably linked by a cyclic bridging group
represented by the general formula II: II 3
[0045] wherein
[0046] Q is a 4- to an 8-membered carbocyclic ring which may be
fully or partially saturated;
[0047] t is an integer from 2 to 16;
[0048] each R group is independently hydrogen, --OH, --CH.sub.2-A,
--OCH.sub.2CH(OH)CH.sub.2-A or a functional group capable of
forming a conjugate with a biomolecule, provided that at least two
of the R groups are selected from --CH.sub.2-A or
--OCH.sub.2CH(OH)CH.sub.2-A; and
[0049] A is the monomer of formula I.
[0050] Of course, the cyclic bridging group may be used to link
known moieties as well. Where it is a known moiety which is being
linked by the cyclic bridging group of formula II, A is any moiety
capable of chelating a metal atom.
[0051] Preferred bridging groups of the formula II are those
wherein 4
[0052] is a compound of the following formula III: 5
[0053] wherein
[0054] each R.sup.1 through R.sup.12 group is independently
hydrogen, --OH, --CH.sub.2-A, --OCH.sub.2CH(OH)CH.sub.2-A or a
functional group capable of forming a conjugate with a
biomolecule;
[0055] at least two of R.sup.1 through R.sup.12 are selected from
--CH.sub.2-A or --OCH.sub.2CH(OH)CH.sub.2-A;
[0056] R.sup.8 and R.sup.9 taken together may additionally form the
group --O--[C(RR)]--O-- where each R is independently hydrogen or
alkyl, or R.sup.8 and R.sup.9 taken together may form 6
[0057] and
[0058] A is a moiety described above.
[0059] When A is a known monomer, for example, A is preferably
7
[0060] wherein
[0061] each R' is independently hydrogen, alkyl, alkoxy,
hydroxyalkyl, aryl, aralkyl or arylalkoxy;
[0062] each R" is hydrogen; and
[0063] each n is 1 or 2;
[0064] or a salt thereof.
[0065] Contrast agents with significantly enhanced relaxivities are
of great interest, not only because they offer improved efficacy at
reduced doses for current clinical applications, but also because
they may provide the sensitivities needed for imaging various
biochemical processes.
[0066] Certain preferred compounds having enhanced relaxivities are
(i.) chelates possessing a stability greater than or equal to
10.sup.15 M.sup.-1 and capable of exhibiting an immobilized
relaxivity between about 60 and 200 mM.sup.-1s.sup.-1/metal atom,
for example between about 70 and 150 mM.sup.-1s.sup.-1/metal atom,
or between about 80 and 100 mM.sup.-1s.sup.-1/metal atom; and (ii.)
multimeric chelates possessing monomer units with a stability
greater than or equal to 10.sup.15 M.sup.-1, and having relaxivity
values (not immobilized) greater than 5 mM.sup.-1s.sup.-1/metal
atom.
[0067] In designing new chelates with these elevated relaxivities,
the inventors have noted that immobilized relaxivity can depend
strongly, for example, on the structure of the chelate. Without
being bound by any particular theory, apparently a mechanism that
involves rigidifying the chelate structures in solution with, for
example, alkyl substitutions on the tetraaza ring of chelates,
especially when the substitution is introduced into the carboxylate
arms of the chelates, affects immobilized relaxivity. In the
Solomon-Bloembergen-Morgan (SBM) model, the electronic relaxation
of a paramagnetic metal complex is viewed as occuring through a
dynamic modulation process about the transient zero-field splitting
(ZFS) of the metal's electronic spin levels. This transient ZFS is
induced by the structural distortion of the metal complex from its
ideal symmetry in solution which is thought to be caused by its
collision with solvent molecules. Alkyl substitution on either the
tetraaza ring or the carboxylate arms of a chelate is thought to
reduce the flexibility of the chelate for structural distortion in
solution. This in turn reduces the magnitude of ZFS, giving an
increased immobilized relaxivity value.
[0068] Water relaxivity is generally determined in aqueous Bis Tris
buffer (pH 7) solutions by the standard inversion-recovery method
(known to those in the art) at 20 MHz and 40.degree. C. (See, e.g.,
X. Xhang, Inorganic Chemistry, 31, 1992, 5597, the entire contents
of which are hereby incorporated by reference.) While a
mathematical description of the relaxation mechanism at the
presence of a paramagnetic species is provided by the classical SBM
equations, it is experimentally difficult to explore the dependence
of relaxivity on structure because the relaxivity values of most
low-molecular weight chelates are often controlled by their rapid
tumbling motions in regular aqueous solutions.
[0069] To eliminate the overriding effect of molecular tumbling in
regular aqueous solutions, the relaxivity of a chelate can be
determined in aqueous sucrose solutions or other media that result
in a reduction of molecular reorientation of any solute. In these
solutions, the relaxivity values approximate, under optimal
conditions, the relaxivities of chelates in biologically
immobilized systems such as those covalently attached to cell
surfaces. Hence, these values in sucrose solutions are defined as
immobilized relaxivity. Immobilized relaxivity is generally
determined under the same conditions as water relaxivity (see
supra) except that the viscosity of the solutions is increased to
80 cp by the addition of solid sucrose to the aqueous solution of
chelate, and the temperature is set at 20.degree. C. In the
calculation of relaxivity, one uses the concentration of the solute
in the aqueous solution (before sucrose is added).
[0070] When used for relative comparison, this method serves as a
simple screening technique for differentiating chelates designed
for efficient biological targeting.
[0071] Thus, the compounds of the invention, including compounds of
the formula I and formula II, and salts thereof, may be complexed
with a paramagnetic metal atom and used as relaxation enhancement
agents for magnetic resonance imaging. These agents, when
administered to a mammalian host (e.g., a human) distribute in
various concentrations to different tissues, and catalyze
relaxation of protons (in the tissues) that have been excited by
the absorption of radiofrequency energy from a magnetic resonance
imager. This acceleration of the rate of relaxation of the excited
protons provides for an image of different contrast when the host
is scanned with a magnetic resonance imager. The magnetic resonance
imager is used to record images at various times, generally either
before and after administration of the agents, or after
administration only, and the differences in the images created by
the agents presence in tissues are used in diagnosis. In proton
magnetic resonance imaging, paramagnetic metal atoms such as
gadolinium(III), and manganese(II), chromium(III) and iron(III)
(all are paramagnetic metal atoms with favorable electronic
properties) are preferred as metals complexed by the ligands of the
invention, including the ligands of formula I and formula II.
Gadolinium(III) is the most preferred complexed metal due to the
fact that it has the highest paramagnetism, it has low toxicity
when complexed to a suitable ligand, and it has high lability of
coordinated water. When the distance between the monomeric
gadolinium chelate units in a complex is at least about 6
angstroms, the complexes tend to be sufficiently stable. Those
compounds of formula II, when complexed with gadolinium (III) ions,
are particularly useful. The distance between the monomeric
gadolinium chelate units in these complexes is generally greater
than 6 angstroms (although in certain circumstances a distance of
4.5 angstroms is sufficient), and the rigid bridges of these
complexes assist in reducing independent motion of the gadolinium
ions.
[0072] The metal-chelating ligands of the present invention can
also be complexed with a lanthanide (atomic number 58 to 71) and
used as chemical shift or magnetic susceptibility agents in
magnetic resonance imaging or in magnetic resonance in vivo
spectroscopy.
[0073] While the above-described uses for the metal-chelating
ligands of the present invention are preferred, those working in
the diagnostic arts will appreciate that the ligands can also be
complexed with the appropriate metals and used as contrast agents
in other imaging techniques such as x-ray imaging, radionuclide
imaging and ultrasound imaging, and in radiotherapy.
[0074] Use in Imaging
[0075] To use the ligands of the present invention for imaging,
they are first complexed with an appropriate metal. This may be
accomplished by methodology known in the art. For example, the
metal can be added to water in the form of an oxide or in the form
of a halide or acetate and treated with an equimolar amount of a
ligand of the present invention. The ligand can be added as an
aqueous solution or suspension. Dilute acid or base can be added
(where appropriate) to maintain a suitable pH. Heating at
temperatures as high as 100.degree. C. for periods of up to 24
hours or more may sometimes be employed to facilitate complexation,
depending on the metal and the chelator, and their
concentrations.
[0076] Pharmaceutically acceptable salts of the metal complexes of
the ligands of this invention are also useful as imaging agents.
They can be prepared by using a base (e.g., an alkali metal
hydroxide, meglumine, arginine or lysine) to neutralize the
above-prepared metal complexes while they are still in solution.
Some of the metal complexes are formally uncharged and do not need
cations as counterions. Such neutral complexes may be preferred in
some situations as intravenously administered x-ray and NMR imaging
agents over charged complexes because they may provide solutions of
greater physiologic tolerance due to their lower osmolality.
[0077] The present invention also provides pharmaceutical
compositions comprising a compound of the invention, including a
compound of the formula I or II, or a salt of one of these
compounds, optionally complexed with a metal, and a
pharmaceutically acceptable vehicle or diluent. The present
invention further provides a method for diagnostic imaging
comprising the steps of administering to a host a compound of the
invention, or a salt thereof, which is complexed with a metal, and
obtaining a diagnostic image, preferably a magnetic resonance
image, of said host.
[0078] Sterile aqueous solutions of the chelate complexes of the
present invention are preferably administered to mammals (e.g.,
humans) orally, intrathecally and, especially, intravenously in
concentrations of about 0.003 to 1.0 molar. The metal complexes of
the present invention may be employed for visualization of various
sites. For example, for the visualization of brain lesions using
magnetic resonance imaging, a gadolinium complex of a ligand of the
invention, including a ligand of the formula I or formula II, may
be administered intravenously at a dose of 0.001 to 0.5 millimoles
of the complex per kilogram of body weight, preferably at a dose of
0.001 to 0.3 millimoles/kilogram. For visualization of the kidneys,
the dose is preferably 0.05 to 0.20 millimoles/kilogram. For
visualization of the heart, the dose is preferably 0.001 to 0.3
millimoles/kilogram. For visualization of the liver, the dose is
preferably 0.001 to 0.3 millimole/kilogram.
[0079] The pH of the formulation of the present metal complexes is
preferably between about 6.0 and 8.0, most preferably between about
6.5 and 7.5. Physiologically acceptable buffers (e.g.,
tris(hydroxymethyl)-aminomethane) and other physiologically
acceptable additives (e.g., stabilizers such as parabens) may also
be present.
[0080] It is also advantageous to employ dual scavenging excipients
such as those described in copending application U.S. Ser. No.
032,763, filed Mar. 15, 1993, entitled "DUAL FUNCTIONING EXCIPIENT
FOR METAL CHELATE CONTRAST AGENTS", incorporated herein by
reference. Those excipients have a general formula corresponding
to:
D.sub.s[D'(L')].sub.t
[0081] wherein D and D' are independently Ca or Zn, L' is an
organic ligand which may be different from, or the same as, the
ligand employed to complex the metal, and s and t are independently
1, 2 or 3.
[0082] As already noted, the present invention further includes
multimeric forms of the compounds of the invention, including those
of formula I and formula II, such as dimers, trimers, tetramers,
etc. Known functional groups and technology are readily useable to
provide such multimers.
[0083] Compounds of the present invention may include those
containing functional group(s) capable of forming a conjugate with
a biomolecule. These compounds are preferably chelates, including a
functional group, capable of exhibiting an immobilized relaxivity
between about 60 and 200 mM.sup.-1s.sup.-1/metal atom, for example
between about 70 and 150 mM.sup.-1s.sup.-1/metal atom, or between
about 80 and 100 mM.sup.-1s.sup.-1/metal atom. Similarly, the
chelates, once conjugated to a biomolecule of size greater than or
equal to about 40,000 daltons, are also preferably capable of
exhibiting a relaxivity between about 60 and 200
mM.sup.-1s.sup.-1/metal atom, for example between about 70 and 150
mM.sup.-1s.sup.-1/metal atom, or between about 80 and 100
mM.sup.-1s.sup.-1/metal atom. Preferred biomolecules are peptides,
polypeptides and oligosaccharides or fragments thereof, although
other biomolecules such as proteins, particularly monoclonal
antibodies, lipids, sugars, alcohols, bile acids, fatty acids,
receptor-binding ligands, amino acids and RNA, DNA or modified
fragments of these may be conjugated to the compounds of the
present invention. For smaller biomolecules, the enhanced
relaxivity afforded by the chelates of this invention may be more
fully realized when the chelate-biomolecule conjugate becomes
immobilized in vivo, such as by binding to a receptor on a cell
surface or by binding to another biomolecule.
[0084] Conjugates where a compound of the invention, or salt and/or
multimer thereof, is linked to a biomolecule such as a protein,
provided by the present invention, are novel, as are metal
complexes and pharmaceutical compositions containing, and methods
of using (e.g., for imaging), the aforementioned conjugates.
Conjugation may be achieved in vitro, such as by use of known
conjugation methodologies, or in situ in a living subject by
administration of a compound containing one or more of the
aforementioned functional groups.
[0085] For linking the compounds of the present invention to a
protein, the R groups may be reacted with a protein to produce a
protein conjugate. Preferred proteins are those in serum, wherein
the compound of the invention is directly injected and the
conjugate is formed in situ. It is understood that other functional
groups, as described above, may be used to link the bifunctional
metal-chelating ligands of this invention to proteins such as
monoclonal antibodies or fragments thereof.
[0086] Compounds of the formula I can generally be prepared as
follows:
[0087] Ligands in which the aza macrocyclic ring carbon atoms are
modified are built de novo from suitable aziridine precursors by
cyclotetramerization.
[0088] Cyclotetramerization of N-benzylaziridine has been reported
in the literature. (See, for example, T. E. Burg and G. R. Hansen,
J. Heterocyclic Chemistry, (1968), 5, 305.) The synthetic approach
to the preparation of S,S,S,S-tetramethyltetra-azacyclododecane (5)
is given in scheme 1: 8
[0089] [S]-N-Benzoylalanine(1) is reduced with diborane to yield
[S]-N-benzylalaninol(2). Under Mitsunobu conditions, compound (2)
affords [S]-N-benzyl-2-methyl aziridine(3). Cyclotetramerization
under p-toluene sulfonic acid catalysis in ethanol, followed by
treatment with ammonium hydroxide (NH.sub.4OH), furnishes
S,S,S,S-tetra-N-benzyl-tetramethyltetra- azacyclododecane(4), which
is debenzylated under transfer hydrogenolytic conditions to obtain
S,S,S,S-tetramethyltetraaza-cyclododecane(5).
[0090] Tetraalkylation of compound (5) with t-butyl bromoacetate in
the presence of sodium carbonate, followed by deprotection with
trifluoroacetic acid and anisole, affords the ligand (6).
[0091] Tetraalkylation of compound (5) with benzyl
2-triflyloxylactate (prepared as described by S. I. Kang et al.,
Inorg. Chem., (1993), 3, 2912-2918) in the presence of sodium
carbonate, followed by catalytic hydrogenolytic debenzylation
furnishes the ligand (7).
[0092] Tris-alkylation of compound (5) with t-butyl bromoacetate in
the presence of sodium bicarbonate, followed by deprotection by
treatment with trifluoroacetic acid and anisole, provides the
ligand (8).
[0093] For preparing multimeric ligands,
1,4,7-tris-carboxymethyl-1,4,7,10- -tetraazacyclododecane-10-yl
(DO3A) units, for example, are attached to a bridging unit by two
different methods. In the first method, a spiro epoxide on the
bridging unit is used to alkylate DO3A. In the second method, a
glycidyloxy moiety is attached to the bridging unit which then is
used to alkylate DO3A.
[0094] By way of example, N-alkylation of DO3A with suitable
epoxides of formula (9) below, wherein X is a carbocyclic, fused
heterocyclic or spiro-heterocyclic unit, and n is 2, 4 or 8,
affords multimeric ligands of the formula (10): 9
[0095] Epoxides of formula (9) are generated by the Prilezhaev
Reaction in which olefins of formula (11) are treated with peracids
such as m-chloroperbenzoic acid: 10
[0096] Olefins of formula (11) are generated by the alkylation of
alcohols of formula (12): 11
[0097] Another mode of attachment of DO3A would be to epoxidize
olefins of formula (13) to obtain epoxides of formula (14), and
then alkylating DO3A with the epoxides to form ligands of formula
(15): 12
[0098] These two method of attachment of DO3A could also be present
in the same bridging unit X leading to structures as in formula
(16): 13
[0099] Representative bridging units are exemplified in formulas
(17), (18), (19), (20), (21) and (22): 14
[0100] The structures shown in formulas (17) to (22) are only by
way of representative examples. They will, in actuality, be a
mixture of all the possible diastereomers, if the attachment is
through a 2-hydroxypropyloxy link. In the case of formula 22, for
example, in addition to the presence of diastereomers, the product
would also consist of the various geometric isomers that could
result when the two myo-inositol molecules are coupled to each
other. The coupling of the two myo-inositol units is achieved by
reacting the compound of formula (23) with cyclohexane-1,4-dione
(24): 15
[0101] The bridging unit for formula 17 is generated from
commercially available diethyl
cyclohexane-1,4-dione-2,5-dicarboxylate by lithium aluminum hydride
reduction as described in J. G. Murphy, J. Med. Chem., (1966), 9,
157.
[0102] The bridging unit for formula (18) is generated by treating
myo-inositol (25) with 2,2-dimethoxypropane in the presence of
p-toluene sulfonic acid as descirbed by Giggs et al., Carbohydrate
Res., 1985, 142, 132: 16
[0103] The bridging unit (19) is made from (18) by acidic
hydrolysis. The unit (20) is prepared from myo-inositol (25) by
reaction with excess 2,2-dimethoxypropane in the presence of
p-toluene-sulfonic acid. The unit (21) is made from the bis-epoxide
precursor to the unit (17) by methods desribed above for
functionalizing hydroxy groups.
[0104] The ligand of Example (10), below, bearing a phosphonomethyl
arm, is made by treating DO3A with phosphorus acid and formaldehyde
as described by M. Tazaki et al., Chem, Lett., 1982, 571.
[0105] All stereoisomers of the compounds and complexes of the
present invention are contemplated herein, whether alone (that is
substantially free of other isomers), in a mixture of certain
stereoisomers (for example, as a racemate) or in any other mixture
thereof.
[0106] The invention will now be further described by the following
examples. In the structures of examples 1-6, R is 17
Example 1
[0107]
(1.alpha.,2.beta.,4.beta.,5.beta.)-10,10'-[(1,2,4,5-Tetrahydroxy-1,-
4-Cyclohexanediyl)bis(Methylene)]bis[1,4,7,10-Tetraazacyclododecane-1,4,7--
Triacetic Acid], Digadolinium Salt 18
[0108] A. Trans-2,5-Dihydroxy-1,4-Dimethylene Cyclohexane
[0109] A warm solution of dry, recrystallized diethyl
1,4-cyclohexanedione-2,5-dicarboxylate (30.75 g, 120 mmol) in 350
ml anhydrous tetrahydrofuran (THF) was added to a refluxing
solution of 1.0 M LAH in THF (600 mL) over a 2 hour period. After
an additional 1.5 hours at reflux and subsequent cooling, a
saturated solution of aqueous Rochelles's salt (app. 73 mL) was
added dropwise to the reaction mixture. The tartrate complex was
filtered, washed with THF and the filtrate was concentrated to a
paste. The paste was recrystallized from hot ethyl acetate; yield
4.39 g (31.3 mmol, 26.1%). A second recrystallization yielded 3.81
g (27.2 mmol, 22.6%). M.P. : 160-161.5.degree. C., uncorrected
(lit. 162.5 -163.degree. C.).
[0110] B.
(3.alpha.,4.alpha.,6.beta.,9.beta.)-1,7-Dioxadispiro[2.2.2.2]-De-
cane-4,9-Diol
[0111] A solution of wet, 80% m-chloroperoxybenzoic acid (mCPBA,
7.00 g, app. 30 mmol) in 30 mL dry dichloromethane
(CH.sub.2Cl.sub.2) was dried over anhydrous magnesium sulfate
(MgSO.sub.4). This solution was added to a solution of dry Compound
A (1.40 g, 10.0 mmol) in 15 mL dry CH.sub.2Cl.sub.2). The mixture
was stirred at room temperature under a dry nitrogen atmosphere for
16 hours. A second sample (550 mg) of mCPBA was added to ensure
complete conversion of the dimethylene compound to the diepoxide.
After 3 hours, the mixture was evaporated to dryness and excess
mCPBA/m-chlorobenzoic acid was removed by treating the residue with
diethyl ether. The ether-insoluble material was filtered, washed
with ethyl ether (Et.sub.2O) and air dried to yield 1.35 g of crude
Compound B. The crude material was recrystallized from a minimum of
hot methanol; yield 967 mg (5.6 mmol, 56.1%). M.P.: 231-233.degree.
C.
[0112] C.
(1.alpha.,2.alpha.,4.beta.,5.beta.)-10,10'-[(1,2,4,5-Tetrahydrox-
y-1,4-Cyclohexanediyl)bis(Methylene)]bis[1,4,7,10-Tetraazacyclododecane-1,-
4,7-Triacetic Acid], Hexasodium Salt
[0113] The pH of a suspension of
1,4,7,10-tetra-azacyclododecane-1,4,7-tri- acetic acid
(DO3A)-H.sub.2SO.sub.4 (prepared as described in D. D. Dischino et
al., Inorg. Chem., 1991, 30, 1265-69; 2.67 g, 6.0 mmol) in 2.0 mL
water was adjusted to approximately pH 12.5 with 5.40 mL 5N sodium
hydroxide. This solution was warmed to 50.degree. C., and Compound
B (517 mg, 3.0 mmol) was added portion-wise over 5.5 hours.
Crystals developed overnight from the warm mixture. After cooling,
the crude crystals (3.4 g) were isolated and were recrystallized
from 20 mL hot water to yield 1.3 g (1.4 mmol, 45%) of Compound C.
Mass Spectrum (FAB): (M-7H+8 Na).sup.+ at 1040; (M-6H+7 Na).sup.+
at 1019; (M-5H+6 Na).sup.+ at 997; (M-4H+5 Na).sup.+ at 975;
(M-3H+4 Na).sup.+ at 953; (M-2H+3 Na).sup.+ at 931. Elemental
analysis: Found: C 42.53, H 6.10, N 10.90. Calculated for:
C.sub.36H.sub.58N.sub.8Na.sub.6O.sub.16.multidot.1.16 H.sub.2O: C
42.49, H 5.97, N 11.01.
[0114] D.
(1.alpha.,2.alpha.,4.beta.,5.beta.)-10,10'-[1,2,4,5-Tetrahydroxy-
-1,4-Cyclcohexanediyl)bis(Methylene)]bis[1,4,7,10-Tetraazacyclododecane-1,-
4,7-Triacetic Acid], Digadolinium Salt
[0115] The pH of a solution of Compound C (760 mg, 762 .mu.mol) in
5.0 mL deionized water was adjusted to pH 4.83 with 225 .mu.L
acetic acid (HOAc). A solution of Gd(OAc)3.multidot.4 H.sub.2O (680
mg, 1.67 mmol) in 5.0 mL warm deionized water was added to the
ligand solution over a 10 minute period; final pH 4.73. After 1
minute of stirring at room temperature, a precipitate began to
develop. The volume of the precipitate increased with time and the
reaction was left to run overnight. The precipitate was filtered,
extensively washed with water, and dried; yield 741 mg (631
.mu.mol, 83%). An analytical sample was prepared by
recrystallization from hot water. Mass spectrum: (FAB): (M+H).sup.+
at 1169 through 1177. Elemental analysis: Found: C 35.93, H 5.50, N
8.84. Calculated for C.sub.36H.sub.58Gd.sub.2N.sub.8O.sub.16.mult-
idot.2.5 H.sub.2O: C 35.49, H 5.21, N 9.20.
Example 2
[0116] (3a.alpha.,4.alpha., 5.beta.,6.alpha.,7.beta.,
7a.alpha.)-10,10'10",10"'[[Hexahydro-2,2-Dimethyl-1,3-Benzodioxol-4,5,6,7-
-Tetrayl]Tetra(Oxy)-Tetra(2-Hydroxy-3,1-Propanediyl)]Tetrakis[1,4,7,10-Tet-
raazacyclododecane-1,4,7-Triacetic Acid], Tetragadolinium Salt
19
[0117] A. (+)-1.2-O-Isopropylidene-Myo-Inositol
[0118] A mixture of myo-inositol (25.0 g, 140 mmol),
2,2-dimethoxypropane (42.5 mL, 350 mmol) and p-toluenesulfonic acid
(250 mg) in dimethylsulfoxide (80 mL) was stirred at 100.degree. C.
for 45 minutes until the solution became homogenous. After stirring
the mixture at ambient temperature for 30 minutes, triethylamine
(2.5 mL), ethanol (100 mL) and ether (500 mL) were introduced
causing precipitation. The solid material was filtered, washed with
ether-methanol (200 mL, 5:1) and ether, and dried to obtain the
ketal compound A (9.72 g) as a white solid in 32% yield. m.p.:
182.4-182.9.degree. C.
[0119] B. (3a.alpha.,4.alpha.,5.beta.,
6.alpha.,7a.alpha.,)-Hexahydro-2,2--
Dimethyl-Tetra-Kis-4,5,6,7-(2-Propenyl-Oxy)-1,3-Benzodioxole
[0120] To a suspension of sodium hydride (545 mmol) in
dimethylformamide (50 mL) was added the ketal compound A (20.0 g,
90.8 mmol) dimethylformamide (20 mL) at ambient temperature under
nitrogen. After stirring the suspension for 2 hours, allyl bromide
(83.4 g, 689 mmol) in dimethylformamide (50 mL) was reacted for 18
hours. The unreacted sodium hydride was carefully quenched with
water, and then the solvent was removed to obtain a dark brown oil.
The residue was partitioned in water (100 mL) and dichloromethane
(100 mL) and extracted with dichloromethane (100 mL.times.3). The
combined organic layers were dried over sodium bisulfate and
evaporated to obtain a brownish liquid (about 45 mL). The crude
product was purified by silica gel flash chromatography (ethyl
acetate and hexanes, 1:8) to afford compound B (26.2 g) as a pale
yellow liquid in 76% yield.
[0121] C.
(3a.alpha.,4.alpha.,5.beta.,6.alpha.,7a.alpha.)-Hexahydro-2,2-Di-
methyl-Tetra-Kis-4,5,6,7-(Oxiranylmethoxy)-1,3-Benzodioxole
[0122] To compound B (6.0 g, 15.8 mmol) in dichloromethane (10 mL)
was added dropwise m-chloroperbenzoic acid (85% from ICN, 19.2 g,
94.6 mmol) dissolved in dichloromethane (100 mL). A homogeneous
solution was initially obtained but a white solid, presumed to be
m-chlorobenzoic acid, slowly precipitated out of the solution after
about 15 minutes. After 24 hours, sodium metabisulfite (100 mL, 5%
in water) and sodium bicarbonate (200 mL, 10% in water) were added
and the resulting solution was extracted with dichloromethane (100
mL.times.3). The organic layers were combined and dried over sodium
sulfate. The residue, obtained from removal of the solvent, was
purified by flash silica gel chromatography to obtain compound C as
a colorless liquid in 76% yield.
[0123] D. (3a.alpha.,4.alpha.,5.beta., 6.alpha.,
7.beta.,7a.alpha.,)-10,10-
',10",10'"-[[Hexahydro-2,2-Dimethyl-1,3-Benzodioxol-4,5,6,7-Tetrayl]-Tetra-
(Oxy)Tetra(2-Hydroxy-3,1-Propanediyl)]Tetrakis[1,4,7,10-Tetraazacyclo-Dode-
cane-1,4,7-Triacetic Acid], Triethylamine (1:4) Salt
[0124] To a suspension of DO3A (9.77 g, 28.2 mmol) dissolved in
alkaline water (6 mL, 10 N sodium hydroxide was used to obtain a pH
10 solution) at 40-50.degree. C. was added the tetraepoxide
compound C (1.54 g, 3.46 mmol) in acetonitrile (5 mL). The reaction
mixture was stirred for 2 days maintaining the same pH. The crude
product was purified by DEAE Sephadex chromatography using
triethylammonium bicarbonate (pH 7.5) as the eluent. The buffer
concentration to bring out the product compound D was 200 to 500
mM. Removal of the buffer gave compound D (7.8 g) in 58% yield.
m.p.: 178.degree. C. (decomp.)
[0125] E.
(3a.alpha.,4.alpha.,5.beta.,6.alpha.,7.beta.,7a.alpha.)-10,10'10-
"10"'-[[Hexahydro-2,2-Dimethyl-1,3-Benzodioxol-4,5,6,7-Tetrayl]Tetra(Oxy)T-
etra(2-Hydroxy-3,1-Propanediyl)]Tetrakis[1,4,7,10-Tetraazacyclododecane-1,-
4,7-Triacetic Acid], Tetragadolinium Salt
[0126] To compound D (216 mg, 96 .mu.mol) dissolved in water (1 mL)
was added gadolinium acetate (236 mg, 580 .mu.mol) dissolved in
water (2 mL). The solution was heated to 65.degree. C. for 18
hours. The pH was maintained between 4.0 and 6.0 during the
chelation reaction. The material was loaded to a CHP20P column and
eluted with.water (1.9 L) followed by 10% ethanolic water (1 L).
The product was eluted with 10% ethanol-containing fraction, as
identified by HPLC, and evaporated to dryness to yield the title
compound (283 mg) as a white crystalline solid in a quantitative
yield. MS (FAB): m/z: 2447.8 [(M+H).sup.+, base peak]. Analysis
Calculated for C.sub.77H.sub.124N.sub.16O.sub.34Gd.sub.4.multido-
t.11.41H.sub.2O: C, 34.87; H, 5.58; N, 8.45. Found: C, 34.68; H,
5.98; N, 8.30; H.sub.2O, 7.75 (desorprtion KF).
Example 3
[0127]
3,4,5,6-Tetra-O-[2-Hydroxy-3-[4,7,10-Tris(Carboxymethyl)-1,4,7,10-T-
etraazacyclododecan-1-yl]propyl]-Myo-Inositol, Tetragadolinium Salt
20
[0128] A.
3,4,5,6-Tetra-O-[2-Hydroxy-3-[4,7,10-Tris-(Carboxymethyl)-1,4,7,-
10-Tetraazacyclo-Dodecan-1-yl]Propyl]-Myo-Inositol, Tetratriethyl
Ammonium Salt
[0129] Compound D from Example 2 was treated with 1.0 N aqueous
hydrochloric acid (10 mL) for 0.5 hours to remove the ketal group.
The resulting-solution was applied to a polyvinylpyridine (PVP)
column (2.5.times.30 cm) and eluted with water. Silver nitrate was
given to test each fraction to detect any breakage of chloride ion.
Removal of the water from the fractions containing the product gave
the title compound (910 mg) in 80% yield. m.p.: 210.degree. C.
(decomp). MS (FAB): m/z: 1789.9 [(M+H).sup.+, base peak]; 1811.9
[(M+Na).sup.+]. Analysis Calculated for
C.sub.74H.sub.132N.sub.16O34.multidot.11.0H.sub.2O: C, 44.63; H,
7.81; N, 11.27. Found: C, 44.63; H, 7.84; N, 11.41; H.sub.2O, 6.43
(desorption KF); ROI, 0.17.
[0130] B.
3,4,5,6-Tetra-O-[2-Hydroxy-3-[4,7,10-Tris(Carboxymethyl)-1,4,7,1-
0-Tetraazacyclo-Dodecan-1-yl]Propyl]-Myo-Inositol, Tetragadolinium
Salt
[0131] To gadolinium acetate (595 mg, 1.46 mmol) dissolved in water
(1.5 mL) was added Compound A (550 mg, 0.246 mmol) dissolved in
water (2 mL). The chelation mixture was kept overnight at
60.degree. C. and the pH of the solution maintained between 4.0 and
5.0. The resulting mixture was loaded to a CHP20P column
(2.5.times.25 cm) and eluted with water followed by 10% ethanol.
The title compound was brought out by 10% ethanolic water. Removal
of the solvents from the fractions containing the product gave the
title compound as a white solid in 93% yield. MS (FAB):
2408.7[(M+H).sup.+, .sup.158Gd)]: Analysis Calculated for
C.sub.74H.sub.120N.sub.16O.sub.34Gd.sub.4.multidot.4.86 H.sub.2O:
C, 35.63; H, 5.24; N, 8.98. Found: C, 35.48; H, 5.74; N, 8.75; 3.51
H.sub.2O (desorption KF); ROI, 31.52.
Example 4
[0132]
(1.alpha.,2.alpha.,3.alpha.,4.beta.,5.alpha.,6.beta.)-10,10'-[(2,3,-
5,6-Tetrahydroxy-1,4-Cyclohexanediyl)bis(2-Hydroxy-3,1-Propanediyl)]-bis[1-
,4,7-10-Tetraazacyclododecane-1,4,7-Triacetic Acid], Digadolinium
Salt 21
[0133] A.
[3aR-(3a.alpha.,4.alpha.,4a.alpha.,7a.alpha.,8.beta.,8b)]-Hexahy-
dro-2,2,6,6-Tetramethyl-4,8-bis-(2-Propenyl-Oxy)Benzo[1,2-d:4,5-d']bis
[1,3]Dioxole
[0134] A sample of sodium hydride (NaH)(8.31 g, 156 mmol; 45% in
mineral oil) was added to an ice cold solution of dry
1,2:4,5-di-O-isopropylidene- -myo-inositol (10.00 g, 38.4 mmol,
prepared as indicated in Gigg et al., Carbohyd. Res., 1985, 142,
132) in dry dimethylformamide (DMF) (80 ml). After stirring for 0.5
hours at room temperature, allyl bromide (8.31 mL, 96 mmol) was
added dropwise over 5 minutes. During the addition, the temperature
was maintained at 25.degree. C. by cooling in a cold water bath.
After 1.5 hours, the reaction was stopped by the careful addition
of water and the mixture was evaporated to dryness in vacuo. The
residue was dissolved in water, extracted with ethyl acetate
(EtOAc) (4.times.50 ml), and washed with water (2.times.20 ml) and
brine (1.times.20 ml). The organic layer was dried over sodium
sulfate (Na.sub.2SO.sub.4) and evaporated to dryness under reduced
pressure. The residue was recrystallized from hexane to give pure
product as a colorless solid (12.88 g, yield 98.5%). M.P.
82-83.degree. C., uncorrected.
[0135] B.
(3a.alpha.,4.alpha.,4a.alpha.,7a.alpha.,8.beta.,8b)-Hexahydro-2,-
2,6,6-Tetramethyl-4,8-bis(Oxiranylmethoxy)Benzo
[1,2-d:4,5-d']bis[1,3]Diox- ole
[0136] A solution of 80% mCPBA (13.09 g, 60.7 mmol) in dry
dichloromethane (CH.sub.2Cl.sub.2)(95 ml) was added to a cooled
solution containing Compound A (5.11 g, 15.0 mmol) in dry
CH.sub.2Cl.sub.2 (15 ml). After stirring at room temperature
overnight, the mixture was treated with saturated aqueous sodium
sulfite (Na.sub.2SO.sub.3) followed by saturated aqueous sodium
bicarbonate (NaHCO.sub.3) until the aqueous layer was neutral. The
organic layer was dried over sodium sulfate (Na.sub.2SO.sub.4)
followed by evaporation to dryness in vacuo. The residue was
recrystallized from hot ethanol to give the pure product (4.05 g,
yield 72.4%). M.P. 103-105.degree. C. (uncorrected)
[0137] C. (1.alpha.,2.alpha.,3.alpha.,4.beta.,
5.alpha.,6.beta.)-10,
10'-[(2,3,5,6-Tetra-Hydroxy-1,4-Cyclohexanediyl)bis(2-Hydroxy-3,1-Propane-
diyl)]bis[1,4,7-10-Tetraazacyclodode-Cane-1,4,7-Triacetic Acid],
Triethylamine (1:2) Salt
[0138] A sample of crude
(3a.alpha.,5.alpha.,5.beta.,6.alpha.,7.beta.,7a.a-
lpha.)-10-10'-[[hexahydro-2,2-dimethylbenzol[1,2-d:4,5-d']-bis[1,3]dioxol--
4,8-diyl]di(oxy)di(2-hydroxy-3,1-propanediyl)]bis[1,4,7,10-tetraazacyclodo-
decane-1,4,7-triacetic acid] was prepared by heating a solution of
DO3A.multidot.H.sub.2SO.sub.4 (28.24 g, 63.5 mmol) and Compound B
(5.92 g, 15.9 mmol) in 10 M sodium hydroxide (NaOH) (28.0 mL),
H.sub.2O (20 mL) and 1,4-dioxane (17 mL) at 50.degree. C. for 70
hours. The pH of the crude mixture was adjusted to pH 1 using 1N
hydrochloric acid (HCl) and was heated at 55.degree. C. for 1.5
hours. After the hydrolysis was complete, the pH of the solution
was adjusted to 9 with dilute NaOH and the mixture was applied to a
DEAE-Sephadex A-25 column (HCO.sub.3.sup.- form, 2L). The column
was washed with water, and the product was eluted with a linear
gradient of 5-250 mM TEAB buffer (pH 7.5, 4 L each). The fractions
which contained pure Compound C were combined, freed of TEAB, and
lyophilized to give Compound C as the di(triethylammonium) salt
(18.22 g, yield 97.2%). The mass spectral analysis of this product
indicated the presence of chloride ion. A sample of the
contaminated product (10.00 g) was dissolved in water and was
applied onto a AG50W.times.8 column (H.sup.+ form , 500 ml). The
column was washed with water, and the product was eluted with 0.5 M
ammonium hydroxide (NH.sub.4OH). The fractions which contained
Compound C were combined and evaporated to give the ammonium salt
(2.91 g). The ammonium salt was dissolved in water, applied to a
DEAE-Sephadex A-25 column (HCO.sub.3.sup.- form, 2L). The column
was washed with water and the product was eluted with 200 mM TEAB
(pH 7.5, 5L). The fractions containing pure material were combined,
freed of TEAB, and lyophilized to give pure Compound C as the
di(triethylammonium) salt (2.86 g, theo. yeild 25.9%).
[0139] D.
(1.alpha.,2.alpha.,3.alpha.,4.beta.,5.alpha.,6.beta.)-10,10'-[(2-
,3,5,6-Tetrahydroxy-1,4-Cyclohexanediyl)]bis(2-Hydroxy-3,1-Propanediyl)]bi-
s[1,4,7,10-Tetraazacyclododecane-1,4,7-Triacetic Acid],
Digadolinium Salt
[0140] A sample of Compound C (1.02 g) was dissolved in a solution
containing Gd(OAc).sub.3.multidot.4 H.sub.2O (1.219 g, 3 mmol) in
water (10 mL) and was heated at 50.degree. C. for 16 hours. The
solution was applied onto a CHP20-P column (500 mL). The salt
contaminants-were eluted with water, and the product was eluted
with 10% ethanol in water. The appropriate fractions were combined
and evaporated to dryness in vacuo. The glassy residue was
dissolved in water, and lyophilized to give pure title compound
(1.04 g, yield 80.5%). MS (FAB): (M+H).sup.+ at 1295.2 (major
isotope). Elemental analysis: Found: C, 36.83; H, 5.63; N, 8.46%.
Calculated for
C.sub.40H.sub.66Gd.sub.2N.sub.8O.sub.20.multidot.1.20 H.sub.2O: C,
36.53; H, 5.24; N, 8.52%.
Example 5
[0141]
10-[[1,4-Dihydroxy-2,5-bis[2-Hydroxy-3-[4,7,10-Tris-(Carboxymethyl)-
-1,4,7,10-Tetraazacyclododecan-1-yl]-Propoxy]-4-[[4,7,10-Tris(Carboxymethy-
l)-1,4,7,10-Tetraazacyclododecan-1-yl]Methyl]Cyclohexyl]Methyl]-1,4,7,10-T-
etraazacyclododecane-1,4,7-Triacetic Acid, Tetragadolinium Salt
22
[0142] A. 1,7-Dioxadispiro [2,2,2,2]-Decane-4,9-Diallyldiol
[0143] To a suspension of sodium hydride (432 mg, 18 mmol) in
anhydrous dimethylformamide (15 ml), a solution of bisepoxydiol
(1.0 g, 6 mmol) (described in Example 1B) in anhydrous
dimethylformamide (60 ml) was added dropwise and the mixture was
stirred at room temperature for 60 minutes. Allyl bromide (2.178 g,
18 mmol) was added and the mixture was stirred at room temperature
for 18 hours. Excess of sodium hydride was decomposed with water (2
ml), dimethylformamide removed in vacuo and the residue extracted
with ethyl acetate (2.times.100 ml), washed with water (2.times.50
ml) and dried. Solvent removal afforded the crude product. Silica
gel (25 g) column chromatography using hexanes and ethyl acetate
(2/1) as the eluent afforded the pure diallyl derivative compound A
as a colorless solid (0.7 g, yield 48%). Melting point:
63.5-65.5.degree. C. (uncorrected).
[0144] B. (3.alpha., 4.alpha., 6.beta.,
9.beta.)-4,9-bis(Oxiranylmethoxy)--
1,7-Dioxadispiror[2,2,2,2]Decane
[0145] A solution of m-chloroperoxybenzoic acid (4.3 g, 80%, 18.5
mmol) in dichloromethane (30 ml) was dried over anhydrous magnesium
sulfate. This solution was added dropwise to a solution of compound
A (1.25 g, 5 mmol) in dry dichloromethane (25 ml). The reaction
mixture was stirred at room temperature for 20 hours. Excess of
m-chloroperoxybenzoic acid was decomposed with saturated aqueous
sodium metabisulfate solution (15 ml). The organic layer was
separated, washed with saturated sodium bicarbonate (2.times.50
ml), and with water (2.times.50 ml). The organic layer was dried
over magnesium sulfate and evaporated to dryness. Recrystallization
of the crude material from hot ether afforded the pure tetraepoxide
compound B as white crystals (1.1 g, yield 77.5%). Melting point:
93.0-96.0.degree. C. (uncorrected).
[0146] C.
10-[[1,4-Dihydroxy-2,5-bis[2-Hydroxy-3-[4,7,10-Tris-(Carboxymeth-
yl)-1,4,7-10-Tetraazacyclododecan-1-yl]-Propoxy]-4-[[4,7,10-Tris(Carboxyme-
thyl)-1,4,7,10-Tetraazacyclododecan-1-yl]Methyl]cyclohexyllmethyl]-1,4,7,1-
0-Tetraazacyclododecane-1,4,7-Triacetic Acid, Triethylamine (1:4)
Salt
[0147] A solution of DO3A (17.3 g, 50 mmol) in water was made (80
ml) and the pH of the solution adjusted to 12 with 5 N sodium
hydroxide solution. The solution was heated to 80.degree. C. and a
solution of compound B (1.42 g, 5 mmol) in dioxane (10 ml) was
added dropwise. The mixture was stirred at this temperature for 82
hours. At the end of 82 hours, the pH of the reaction mixture was
adjusted to 7 with acetic acid. The mixture was diluted and loaded
on to a Sephadex G-25 column. The column was eluted first with
100-250 mM triethyl ammonium bicarbonate (TEAB) buffer and then
with 250-500 mM TEAB buffer. Fractions containing the pure
tetrameric material were combined and solvent removal afforded the
pure product as the tetra triethyl ammonium salt of the title
compound as a colorless glassy solid (2.85 g, yield 35%). Mass
Spectrum: 1670 (M+H).sup.+. 102 (CH.sub.3CH.sub.2).sub.3N.sup.+H
Elemental Analysis: Found: C, 53.70; H, 9.15; N, 13.62; H.sub.2O,
0.58%. Calculated for
C.sub.94H.sub.184N.sub.20O.sub.30.multidot.0.67 H.sub.2O: C, 54.11;
H, 8.85; N. 13.42; O, 23.52%.
[0148] D.
10-[[1,4-Dihydroxy-2,5-bis[2-Hydroxy-3-[4,7,10-Tris-(Carboxymeth-
yl)-1,4,7,10-Tetraazacyclododecan-1-yl]-Propoxy]-4-[[4,7,10-Tris(Carboxyme-
thyl)-1,4,7,10-Tetraazacyclododecan-1-yl]Methyl]Cyclohexyl]Methyl]-1,4,7,1-
0-Tetraazacyclododecane-1,4,7-Triacetic Acid, Tetragadolinium
Salt
[0149] A sample of Compound C (0.2 g) was dissolved in 3 mL
deinonized water (reactant I). In a separate vial, 0.18 g of
GdCl.sub.3 powder was dissolved in 5 mL deionized water (reactant
II). At about 70.degree. C. and constant stirring, reactant I was
added into reactant II dropwise over a time period of several
hours. Meanwhile, 5N NaOH was pipetted into the solution from time
to time to neutralize the protons released from chelation and
maintain the reaction pH around 6. The excess metal ions were
precipitated in the form of M(OH).sub.3.multidot..times.H.sub.2O by
raising solution pH to about 9 and incubating at both about
70.degree. C. and room temperature for several hours. The
precipitate was subsequently removed through centrifugation and
filtration with 0.22 .mu.m membrane. The filtrate was finally
condensed and neutralized to pH 7 in preparation for HPLC
purification.
[0150] The crude chelate product was purified by preparative HPLC,
using mobile phase gradient (acetonitrile:water) and silica-based
reverse phase column (YMC C18, 5.mu., 200 A). The solvent was
removed from the fractions containing the product. The residue was
further dried in a vacuum oven at about 70.degree. C. overnight.
The yield of the desired chelate was 70%. Mass Spectrum (FAB,
m/e):(Gd.sup.159+H).sup.+ at 2287.5. Elemental Analysis (C,H,N):
Calculated for C.sub.70H.sub.112N.sub.16O.sub-
.30Gd.sub.4.multidot.7.73 H.sub.2O: C.sub.34.66, H 5.30, N 9.24%.
Found: C 34.26, H 5.36, N 9.08%.
Example 6
[0151]
[3aR-(3a.alpha.,3"a.alpha.,4.beta.,4".beta.,5".alpha.,6.beta.,6"b,7-
.alpha.,-7".alpha.,7a.alpha.,7"a.alpha.)]-Dodecahydro-4,4",5,5",6,6"7,7"-O-
ctakis-[[2-Hydroxy-3-[(4,7,10-Tricarboxymethyl)-1,4,7,10-Tetraazacyclodode-
can-1-yl]Propyl]-Oxy]Dispiro-[1,3-Benzodioxole-2,1'-Cyclo-Hexane-4',2"-[1,-
3]-Benzodioxole], Octagadolinium Salt 23
[0152] A. (+)-3,4,5,6-Tetra-O-Allyl-Myo-Inositol
[0153] Compound B from Example 2 (15.3 g, 44.9 mmol) was dissolved
in methanol (30 mL) and treated with 3N hydrochloric acid (30 mL),
with constant stirring at room temperature for 13 hours. The
reaction mixture was neutralized with saturated sodium bicarbonate
solution and extracted with methylene chloride (50 mL.times.3). The
dichloromethane layers were combined and dried with sodium sulfate,
filtered, evaporated in vacuo and purified by column chromatography
(silicon dioxide, ethyl acetate: hexanes (1:1 v/v)) to obtain 9.1 g
(67%) of the desired product compound A.
[0154] B.
[3aR-(3a.alpha.,3"a.alpha.,4.alpha.,4".beta.,5.beta.,5".alpha.,6-
.alpha.,6".beta.,7.beta.,7".alpha.,7a.alpha.,7"a.alpha.)]-Dodecahydro-4,4"-
,5,-5",6,6",7,7"-Octakis-(2-Propenyloxy)-Dispiro[1,3-Benzo-Dioxole-2,1'-Cy-
clo-Hexane-4',2"-[1,3]-Benzodioxole]
[0155] A mixture of the diol compound A (1.0 g, 2.94 mmol),
1,4-cyclohexanedione (165 mg, 1.47 mmol) and p-toluenesulfonic acid
(55 mg, 0.289 mmol) was heated in toluene (30 mL) at reflux for 19
hours. The crude reaction mixture was purified by flash silica gel
column chromatography (about 200 g) using different eluents of
hexane and ethyl acetate (2:1, 1:1 and 1:2, 500 mL each). Three to
four spots that have the molecular ion peak at 757 (m/z) were
pooled to afford an oil (520 mG) in 43% yield, and the resulting
mixture used as a mixture of the isomers of the expected octa-allyl
bis-ketal product. TLC: R.sub.f 0.45, 0.37 and 0.32 in hexanes and
: acetone (5:1, v/v)
[0156] C.
[3aR-(3a.alpha.,3"a.alpha.,4.beta.,4".beta.,5.alpha.,5".alpha.,6-
.beta.,6"b,7.alpha.,7".alpha.,7a.alpha.,7"a.alpha.)]-Dodecahydro-4,4",5,5"-
,6,6",7,7"-Octakis-(Oxyranylmethoxy)-Dispiro[1,3-Benzodioxole-2,1'-Cyclohe-
xane-4',2"-[1,3]-Benzodioxole]
[0157] The oxidation of the octa-allyl compound B (1.13 g, 1.49
mmole) by m-chloroperoxybenzoic acid (3.0 g, 85%, 17.6 mmole) in
dichloromethane (20 mL) was carried out at ambient temperature for
50 hours. The white solid, which precipitated out, during the
oxidation was identified as m-chlorobenzoic acid by .sup.1H-NMR
(CDCl.sub.3). After removal of the solid by filtration, the
dichloromethane filtrate was treated with sodium metabisulfite and
sodium hydroxide where the final pH of the aqueous layer was 11.7.
The organic layer was dried over sodium sulfate and concentrated in
vacuo. The crude solid product was purified by column
chromatography (flash silica gel, 75 g) to obtain a white solid
(670 mg) in 51% yield.
[0158] D.
(3aR-(3a.alpha.,3".alpha.,4.beta.,4".beta.,5.alpha.,5".alpha.,6.-
beta.,6"b,7.alpha.,7".alpha.,7a.alpha.,7"a.alpha.)]-Dodecahydro-4,4",5,5",-
-6,6",7,7"-Octakis-[[2-Hydroxy-3-[(4,7,10-Tricarboxymethyl)-1,4,7,10-Tetra-
azacyclo-Dodecan-1-yl]Propyl]Oxy]Dispiro-[1,3-Benzodioxole-2,1'-Cyclohexan-
e-4',2"-[1,3]-Benzodioxole]
[0159] The octaepoxide Compound C (200 mg, 235 .mu.mol) in
acetonitrile (0.5 mL) was added to DO3A (2.41 g, 7.04 mmol) in
water (4-5 mL) whose pH was adjusted to 9.6 by 10 N sodium
hydroxide at 60.degree. C. The resulting solution was kept for 48
hours and analyzed by PRP-X 100 HPLC column at desirable intervals.
Two relatively pure fractions were obtained after two DEAE Sephadex
A-25 ion exchange columns. One eluted by 300-400 mM
triethyl-ammonium bicarbonate (TEAB) showed a peak at m/z 3328.0
corresponding to the heptamer, whereas-the other by 400 mM TEAB
turned out to be the expected octamer with a peak at m/z 3656.6
(C.sub.154H.sub.268N.sub.32O.sub.68). .sup.1H-NMR (D.sub.2O): d
1.15 (t, 106H, CH.sub.3CH.sub.2N); 1.7-1.9 (m, methylenes of the
middle cyclohexane, 8H); 3.07 (q, 71H, CH.sub.3CH.sub.2N), 2.8-4.6
(m, 228H, all of methines and methylenes of the octamer ligand
except the triethylamine and bridged cyclohexane). .sup.13C-NMR
(D.sub.2O): 8.10 and 46.29 (CH.sub.3CH.sub.2N of triethyl-amine);
41.88, 46.29, 49.25, 49.54, 49.62, 49.76, 50.04, 55.72, 56.43,
58.69, 172-175 (broad due to 24 carboxylate and/or carboxylic acid
groups). MS (FAB): 3,328 (M+H).sup.+. Analysis Calcualted for
C.sub.154H.sub.268N.sub.32O.sub.68-8[N(C.sub.2H.sub.5).sub-
.3].multidot.17.20H.sub.2O: C, 50.81; H, 8.92; N, 11.73. Found: C,
50.33; H, 8.92; N, 11.75; H.sub.2O, 6.10 (desorption
Karl-Fisher).
[0160] E.
[3aR-(3a.alpha.,3"a.alpha.,4.beta.,4".beta.,5.alpha.,5".alpha.,6-
.beta.,6"b,7.alpha.,7".alpha.,7a.alpha.,7"a.alpha.)]-Dodecahydro-4,4",5,5"-
,6,6",7,7"-Octakis[[2-Hydroxy-3-[(4,7,10-Tricarboxy-Methyl)-1,4,7,10-Tetra-
azacyclododecan-1-yl]Propyl]Oxy]Dispiro-[1,3-Benzodioxole-2,1'-Cyclo-Hexan-
e-4',2"-[1,3]Benzodioxole], Octagadolinium Salt
[0161] Compound D (250 mg, 68.4 .mu.mol as the
octa-triethylammonium salt) in water (5 mL) at pH 6.0 was treated
with tetrahydrated gadolinium acetate (222 mg, 546 .mu.mol) at
60.degree. C. for 5 hours. The pH of the aqueous reaction mixture
was maintained at 7.0.+-.1.0. The resulting solution was then
applied to a CHP-20P column (2.5.times.20 cm). The column was
eluted with water (750 mL), 2.5% (300 mL), 5% (300 mL) and 10% (300
mL) of ethanol. The fractions containing the desired compound,
which were eluted by 10% ethanol, were combined and concentrated in
vacuo to obtain the octameric gadolinium chelate (200 mg) as a
white solid in 60% yield. Analysis Calculated for
C.sub.154H.sub.244N.sub.32O.sub.68Gd.sub.8- .multidot.19.58
H.sub.2O.multidot.2.0 C.sub.2H.sub.5OH: C, 35.57; H, 5.58; N, 8.40.
Found: C, 35.06; H, 6.22; N, 8.42; H.sub.2O, 6.61% (desorption
Karl-Fisher).
Example 7
[0162]
[2S-(2.alpha.,5.alpha.,8.alpha.,11.alpha.,)]-2,5,8,11-Tetramethyl-1-
,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid, Gadolinium
Salt 24
[0163] A. L-2-Benzylamino Propanol
[0164] To a solution of N-benzoyl-L-alanine (29.0 g, 150 mmol) in
tetrahydrofuran (200 ml) at 0.degree. C. was added a
tetrahydrofuran solution of diborane (1 M solution, 800 ml) and the
mixture was refluxed for 18 hours. Excess of diborane was
decomposed with methanol and the solvents were removed under
reduced pressure. The residue was dissolved in methanol (100 ml)
and treated with 6 N hydrochloric acid (100 ml). The mixture was
heated at 70.degree. C. for 12 hours and the solvents were removed
under reduced pressure. The residue was co-evaporated with methanol
(5.times.150 ml) and the product was dissolved in water (50 ml),
basified with 5 N sodium hydroxide to pH 12 and extracted with
ethyl acetate (3.times.200 ml). The combined organic layers were
washed with saturated sodium chloride (150 ml), dried and
concentrated to a final volume of 100 ml. 300 ml of hexane was
added and the solution cooled in the refrigerator overnight. The
white crystalline needles deposited were collected and dried to
afford pure compound A (41.4 g, yield 87%). m.p. 46-48.degree.
C.
[0165] B. (S)-1-Benzyl-2-Methyl-Ethyleneimine
[0166] To a solution of Compound A (33.0 g, 200 mmol) and triphenyl
phosphine (79.66 g, 300 mmol) in ether (500 ml) stirred under
nitrogen in an ice bath, was slowly added diethyl azo-dicarboxylate
(95%, 50 ml, 300 mmol). The solution was stirred at room
temperature for 16 hours. A crystalline precipitate
(triphenylphosphine/diethyl hydrazine carboxylate complex) was
filtered off and washed with hexane/ether (1:1, 200 ml). The ether
solution was extracted with 1 N hydrochloric (2.times.100 ml). The
hydrochloric solution was basified with 5N sodium hydroxide,
extracted with ether (3.times.150 ml), dried and concentrated to
afford the crude aziridine compound B as a yellow oily liquid. This
was further purified by distillation under reduced pressure to
afford pure compound B as a colorless liquid (24.3 g, yield 75%).
b.p. 71-72.degree. C. at 4 mm.
[0167] C.
[2S-(2.alpha.,5.alpha.,8.alpha.,11.alpha.)]-2,5,8,11-Tetramethyl-
-1,4,7,10-Tetrakis(Phenylmethyl)-1,4,7,10-Tetraazacyclododecane
[0168] To a solution of compound B (19.1 g, 130 mmol) in ethanol
(250 ml) was added p-toluenesulfonic acid (PTSA, 1.1 g, 6.5 mmol)
and the mixture was stirred at room temperature for 64 hours. At
the end of this time, an additional 1.1 g (6.5 mmol) of PTSA was
added and the mixture was stirred for 48 hours. Ethanol was removed
in vacuo, and the product was purified by column chromatography
over silica gel (400 g) using methanol as the eluent. Fractions
containing the pure product were combined, and solvent removal
afforded 5.8 g of a salt. This was dissolved in methanol (100 mL)
and basified with concentrated ammonia solution. The precipitated
solid was filtered, dried and recrystallized from absolute ethanol
to afford pure compound C as a colorless microcrystalline solid
(2.8 g, yield 14%). m.p.: 147-148.degree. C.
[0169] D.
[2S-(2.alpha.,5.alpha.,8.alpha.,11.alpha.,)]-2,5,8,11-Tetramethy-
l-1,4,7,10-Tetraazacyclododecane
[0170] To a solution of compound C (2.35 g, 4 mmol) in ethyl
acetate (40 mL) and ethanol (400 mL) was added ammonium formate
(2.52 g) and palladium acetate on Carbon (20%, 2.35 g). The mixture
was stirred under reflux for 16 hours. The solution was filtered to
remove the catalyst and the solvents were removed to afford pure
Compound D as a light yellow solid (840 mg, yield 92%).
[0171] E.
[2S-(2.alpha.,5.alpha.,8.alpha.,11.alpha.)]-2,5,8,11-Tetramethyl-
-1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid
[0172] To a solution of compound D (570 mg, 2.5 mmol) in
acetonitrile (200 mL) was added potassium carbonate (4.0 g) and
t-butyl bromo acetate (2.34 g, 12 mmol). The mixture was stirred at
room temperature for 18 hours. Potassium carbonate was filtered
off, acetonitrile removed in vacuo and the residue purified by
column chromatography over silica gel (50 g) using chloroform and
methanol to afford the tetra butyl ester (1.6 g). This material was
dissolved in trifluoroacetic acid (150 mL), anisole (10 mL) was
added, and the mixture was stirred at room temperature for 16
hours. Trifluoroacetic acid was removed in vacuo and anisole was
removed by co-evaporation with water (6.times.50 mL) to afford the
crude product. The residue was dissolved in water (100 mL) and
purified by anion exchange column chromatography over AG1-X2 resin
(150 mL). The column, after washing with water, was eluted with 1 M
formic acid. The fractions containing the pure product were
combined. Solvent removal afforded the pure product. A small amount
of formic acid that remained in the sample was removed by
co-evaporation with water (5.times.50 mL) to obtain pure title
compound as a colorless glassy solid (550 mg, yield 87%).
[0173] F.
[2S-(2.alpha.,5.alpha.,8.alpha.,11.alpha.,)]-2,5,8,11-Tetramethy-
l-1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid,
Gadolinium Salt
[0174] A sample of Compound E (0.1 g) was dissolved in 8 mL
deionized water. 0.04 mL 5N sodium hyroxide (NaOH) was added to
convert the ligand into the monosodium salt (reactant I). In a
separate vial, 0.08 g of GdCl.sub.3 powder was dissolved in 1 mL
deionized water (reactant II). At about 70.degree. C. and constant
stirring, reactant II was added into reactant I dropwise over a
time period of several hours. Meanwhile, 5N NaOH was pipetted into
the solution from time to time to neutralize the protons released
from chelation and maintain the reaction pH around 6. The excess
metal ions were precipitated in the form of
M(OH).sub.3.multidot.H.sub.2O by raising solution pH to about 9.5
and incubating at both about 70.degree. C. and room temperature for
serveral hours. The precipitate was subsequently removed through
centrifugation and filtration with a 0.22 .mu.m membrane. The
filtrate was finally condensed and neutralized to pH 7 in
preparation for HPLC purification.
[0175] The crude chelate product was purified by preparative HPLC,
using mobile phase gradient (CH.sub.3CN:H.sub.20) and silica-based
reversed phase column (YMC C18, 5.mu.,120 A). The solvent was
removed from fractions containing the desired product. The residue
was dried in a vacuum oven at about 70.degree. C. overnight,
furnishing the chelate in 65% yield. Mass Spectrum (FAB, m/e):
Gd.sub.159+Na).sup.+ at 638. Elemental Analysis (C, H, N):
Calculated for C.sub.2OH.sub.33N.sub.4O.sub- .8GdNa.multidot.5.01
H.sub.2O: C, 33.00; H, 5.96; N, 7.70%. Found: C, 32.94; H, 5.63; N,
7.75%
Example 8
[0176]
[aR-(aR*,a'R*,.alpha."R*,.alpha.'"R*,2S*,5S*,8S*,11S*)]-.alpha.,-.a-
lpha.',.alpha.",a'",2,5,8,11-Octamethyl-1,4,7,10-Tetraazacyclododecane-1,4-
,7,10-Tetraacetic Acid, Gadolinium Salt 25
[0177] A.
[.alpha.R-(aR*,.alpha.'R*,.alpha."R*,a'"R*,2S*,5S*,8S*,11S*)]-.a-
lpha.,-.alpha.',.alpha.",a'",2,5,8,11-Octamethyl-1,4,7,10-Tetraazacyclodod-
ecane-1,4,7,10-Tetraacetic Acid
[0178] To a solution of compound D from Example 7 (570 mg, 2.5
mmol) in acetonitrile (200 ml) was added potassium carbonate
followed by L-benzyl-2-trifluoromethanesufonyloxy-propionate
(prepared fresh from benzyl lactate as described in S. I. Kang et
al., Inorganic Chem., 1993, 32, 2912 -2918)(3.9 g, 12.5 mmol) and
the mixture was stirred at room temperature for 48 hours. Potassium
carbonate was filtered off, acetonitrile removed in vacuo and the
residue purified by column chromatography over silica gel (100 g)
using chloroform and the methanol to afford a tetra benzyl ester
(1.1 g). This material was dissolved in a mixture of ethanol (75
ml) and water (10 ml) and hydrogenated over 10% Pd/C (250 mg) for
18 hours. The catalyst was filtered off and solvent removal
afforded the crude product. This was dissolved in water (100 ml)
and purified by anion exchange column chromatography over AG1-X2
resin (150 ml). The column, after washing with water, was eluted
with a gradient of 0-200 mM formic acid. Fractions containing the
pure product were combined. Solvent removal afforded the pure
product. A small amount of formic acid that remained in the sample
was removed by co-evaporation with water (5.times.50 ml) to obtain
pure Compound A as a colorless glassy solid (340 mg, yield
26%).
[0179] B.
[.alpha.R-(aR*,.alpha.'R*,.alpha."R*,.alpha.'"R*,2S*,5S*,8S*,11S-
*)]-.alpha.,-.alpha.',.alpha.",a'",2,5,8,11-Octamethyl-1,4,7,10-Tetraazacy-
clododecane-1,4,7,10-Tetraacetic Acid, Gadolinium Salt
[0180] A sample of Compound A (0.15 g) was dissolved in 8 mL
deionized water. 0.056 mL 5N NaOH was added to convert the ligand
into the monosodium salt (reactant I). In a separate vial, 0.11 g
of GdCl.sub.3 powder was dissolved in 1 mL deionized water
(reactant II). Reactants I and II were treated as described in the
method of Example 7F to yield 74% of the title compound. Mass
Spectrum (FAB, m/e): (Gd.sup.159+Na).sup.+ at 694. Elemental
alalysis: Calculated for C.sub.24H.sub.40N.sub.4O.sub.8GdN-
a.multidot.1.98 H.sub.2O: C, 39.57, H, 6.08, N, 7.69%. Found: C,
39.31, H, 6.19, N, 7.60%.
Example 9
[0181] [2S- (2R*, 5R*, 8R*,
11R*)]-2,5,8,11,-Tetramethyl-1,4,7,10-Tetraaza-
cylcododecane-1,4,7-Triacetic Acid, Gadolinium Salt 26
[0182] A. [2S-(2R*, 5R*,
8R*,11R*)]-2,5,8,11-Tetramethyl-1,4,7,10-Tetraaza-
cyclododecane-1,4,7-Triacetic Acid
[0183] To a solution of Compound D (456 mg, 2 mmol) in acetonitrile
(150 mL) was added sodium bicarbonate (4.0 g) and t-butyl bromo
acetate (2.34 g, 12 mmol), and the mixture was stirred at room
temperature for 48 hours. Sodium bicarbonate was filtered off,
acetonitrile was removed in vacuo and the residue was dissolved in
TFA (25 mL). Anisole (1 mL) was added and the mixture was stirred
at room temperature for 12 hours. TFA was removed in vacuo and
anisole was removed by co-evaporation with water (5.times.50 mL) to
afford the crude product. The residue was dissolved in water (60
mL) and purified by anion exchange column chromatography over
AG1-X2 resin (100 mL). The column, after washing with water, was
eluted with a gradient of water to 50 mm M formic acid. The
fractions were analyzed by HPLC and those containing the pure
product were combined. Solvent removal afforded Compound A. A small
amount of formic acid that remained in the sample was removed by
co-evaporation with water (5.times.50 mL) to obtain pure Compound E
as a colorless glassy solid (320 mg, yield 40%).
[0184] B.
[2S-(2R*,5R*,8R*,11R*)]-2,5,8,11,-Tetramethyl-1,4,7,10-Tetraazac-
ylcododecane-1,4,7-Triacetic Acid, Gadolinium Salt
[0185] A sample of Compound A (0.125 g) was dissolved in 9 mL
deionized water (reactant I). In a separate vial, 0.129 g of
GdCl.sub.3 powder was dissolved in 1.5 mL deionized water (reactant
II). Reactants I and II were treated as described in the method of
Example 7F to yield 80% of the title compound. Mass Spectrum (FAB,
m/e): (Gd.sup.159+H).sup.+ at 559. Elemental Analysis (C,H,N):
Calculated for C.sub.18H.sub.31N.sub.4O.sub.6- Gd.multidot.2.81
H.sub.2O: C 35.60, H 6.08, N 9.22%. Found: C 35.65, H 6.11, N
9.02%.
Example 10
[0186]
10-(Phosphonomethyl)-1,4,7,10-Tetraaza-Cyclododecane-1,4,7-Triaceti-
c Acid, Gadolinium Salt 27
[0187] A.
10-(Phosphonomethyl)-1,4,7,10-Tetraazacyclo-Dodecane-1,4,7-Triac-
etic Acid
[0188] A mixture of DO3A (lg, 2.89 mmol), phosphorus acid (0.485 g,
5.91 mmol), concentrated hydrochloric acid (1.1 mL, 13.4 mmol) and
formaldehyde (1.35 mL of 37% aqueous solution, 16.4 mmol) in water
(2.5 mL) was refluxed for 27.5 hours. Removal of water from the
reaction mixture in vacuo gave an off-white solid (1.74 g) as a
crude product of Compound A. The solid was dissolved in water (50
mL), and the pH of the resulting solution was adjusted to 5.0 by
addition of 1.0 N sodium hydroxide. The aqueous solution was
applied to a strong cation exchange column of AG50WX8 (250 mL,
flowrate, 12.5 mL/minute). Initially, water was used to remove any
negatively charged inorganic species, and ammonium hydroxide (1.0
M) was employed to bring out the crude product. After removal of
the ammonium hydroxide from the fractions containing the product,
the residue dissolved in 5 mM TEAB (pH 7.5) was applied to a DEAE
Sephadex ion exchange column (750 mL, flowrate, 4 mL/minute). The
column was eluted with TEAB, whose concentration varied from 5 to
400 mM. The concentration of the buffer was doubled at every column
volume (1L): 5, 10, 20, 40, 67, 80, 100, 125, 200 and 400 mM.
Compound A required 400 mM TEAB to be brought out from the column.
Evaporation of the buffer from the fractions containing Compound A
gave a white residue (0.9 g). The solid, dissolved in water (35 mL)
was loaded on a strongly basic anion column of Amberlite IRA 900 C
(200 mL, flowrate, 14 mL/minute). Water was first used to remove
triethylamine followed by sulfuric acid (1M) to elute Compound A
from the column. The fractions containing the product were combined
and applied to a column of Reillex 425 poly(4-vinyl)pyridine (PVP)
250 mL, 10 mL/minute). Water was used to elute the product free
from sulfuric acid. Removal of water from the eluate (3.7 L)
yielded Compound A (0.6 g, 47%) as a dense solid.
[0189] B.
10-(Phosphonomethyl)-1,4,7,10-Tetraaza-Cyclododecane-1,4,7-Triac-
etic Acid, Gadolinium Salt
[0190] A sample of Compound A (0.2 g) was dissolved in 5 mL
deionized water and its pH was adjusted to 4 with dilute NaOH.
0.096 g of Gd.sub.2O.sub.3 powder was added slowly. The solution
was refluxed overnight. The excess metal ions were precipitated in
the form of M(OH).sub.3.multidot..times.H.sub.2O by raising
solution pH to about 9.5 with 1N NaOH and incubating at both about
70.degree. C. and room temperature for several hours. The
precipitate was subsequently removed through centrifugation and
filtration with 0.22 .mu.m membrane. The filtrate was finally
condensed and neutralized to pH 7 with 1N HCl in preparation for
HPLC purification. HPLC purification was performed as in Example 7F
to yield 64% of the title compound. Mass Spectrum (FAB, m/e):
(Gd.sup.159+2 Na--H).sup.+ at 640. Elemental Analysis (C,H,N):
Calculated for
C.sub.15H.sub.24N.sub.4O.sub.9PGdNa.sub.2.multidot.2.59 H.sub.2O: C
26.29, H 4.29, N 8.18%. Found: C 26.23, H 4.18, N 7.93%.
* * * * *