U.S. patent application number 09/913711 was filed with the patent office on 2003-01-16 for chelating compounds, their complexes with paramagnetic metals.
Invention is credited to Anelli, Pier Lucio, Beltrami, Andrea, Calabi, Luisella, Franzini, Maurizio, Maiocchi, Alessandro, Ramalingam, Kondareddiar, Ranganathan, Ramachandran S., Virtuani, Mario.
Application Number | 20030013859 09/913711 |
Document ID | / |
Family ID | 11384156 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030013859 |
Kind Code |
A1 |
Franzini, Maurizio ; et
al. |
January 16, 2003 |
CHELATING COMPOUNDS, THEIR COMPLEXES WITH PARAMAGNETIC METALS
Abstract
Compounds able to chelate bi- and trivalent paramagnetic metal
ions, their chelated complexes with said metal ions and the
physiologically compatible salts thereof, as well as the use
thereof as contrast agents in the technique known as "Magnetic
Resonance Imaging" (M.R.I.).
Inventors: |
Franzini, Maurizio; (Milano,
IT) ; Beltrami, Andrea; (Milano, IT) ; Calabi,
Luisella; (Milano, IT) ; Maiocchi, Alessandro;
(Milano, IT) ; Virtuani, Mario; (Milano, IT)
; Anelli, Pier Lucio; (Milano, IT) ; Ramalingam,
Kondareddiar; (Dayton, NJ) ; Ranganathan,
Ramachandran S.; (Princeton, NJ) |
Correspondence
Address: |
Nixon & Vanderhye
1100 North Glebe Road 8th Floor
Arlington
VA
22201-4714
US
|
Family ID: |
11384156 |
Appl. No.: |
09/913711 |
Filed: |
September 24, 2001 |
PCT Filed: |
December 20, 2000 |
PCT NO: |
PCT/EP00/12977 |
Current U.S.
Class: |
534/15 ;
556/138 |
Current CPC
Class: |
A61K 49/085 20130101;
A61K 49/103 20130101; Y10S 977/93 20130101; A61K 49/0438 20130101;
C07J 51/00 20130101; C07F 9/5728 20130101; A61K 49/10 20130101;
C07F 9/3808 20130101 |
Class at
Publication: |
534/15 ;
556/138 |
International
Class: |
C07F 015/02; C07F
005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 1999 |
IT |
MI99A002656 |
Claims
1. Compounds of formula (I), both in the racemic form and optically
active, 27wherein Y is a COOH group or a PO(OH).sub.2 group, with
the proviso that at least one Y group is .dbd.PO(OH).sub.2, R is a
hydrogen atom, or --(CH.sub.2).sub.m13 O--R.sub.2,
(C.sub.1-C.sub.5)-alkyl-(C.sub.- 6-C.sub.10)-aryl or
(C.sub.1-C.sub.5)-alkyl-heteroaryl whose aryl or heteroaryl moiety
comprises 1 or 2 fused rings optionally substituted with one or
more halogen atoms, OH groups, alkyl(C.sub.1-C.sub.5) groups and/or
an OR.sub.3 group, wherein R.sub.2 is (C.sub.1-C.sub.5)-alkyl-(C.s-
ub.6-C.sub.10)-aryl, optionally substituted with one or more
halogen atoms, OH and (C.sub.1-C.sub.5)-alkyl groups; R.sub.3 is
(C.sub.6-C.sub.10) aryl optionally substituted with one or more
halogen atoms, OH and/or (C.sub.1-C.sub.5)-alkyl groups; m ranges
from 1 to 5; R.sub.1 can have the same meanings as R with the
proviso that when Y is PO(OH).sub.2, R.sub.1 is selected from H,
(CH.sub.2).sub.mNH.sub.2, or (CH.sub.2).sub.mCOOH or an amido
derivative thereof, the chelated complexes of said compounds of
formula (I) with the ions of metal elements having atomic number
ranging between 20 and 31, 39, 42, 43, 44, 49, and between 57 and
83, and the salts thereof with physiologically acceptable organic
bases selected from primary, secondary, tertiary amines or basic
amino acids, or with inorganic bases whose cations are selected
from sodium, potassium, magnesium, calcium or mixtures thereof.
2. Compounds as claimed in claim 1 of formula (II) 28wherein R and
R.sub.1 have the meanings defined above.
3. Compounds as claimed in claim 1 of formula (III) 29wherein
R.sub.1 has the meanings defined above.
4. Compounds as claimed in claim 1 of formula (IV), 30wherein R has
the meanings defined above.
5. Compounds as claimed in any of the claims from 1 to 4 wherein R
or R.sub.2 is a (C.sub.1-C.sub.5)-alkyl-(C.sub.6-C.sub.10)-aryl
selected from benzyl, phenetyl, or naphthylmethyl, wherein the aryl
moiety is optionally substituted with one or more halogen atoms or
OR.sub.3 groups wherein R.sub.3 is as defined above.
6. Compounds as claimed in any of the claims from 1 to 4 wherein R
is a (C.sub.1-C.sub.5)-alkyl-heteroaryl selected from pyridylmethyl
or indolylmethyl.
7. A compound as claimed in any of the claims from 1 to 6 selected
from:
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxymethyl-L-pheny-
lalanine]; [4S-(4R*,
12R*)]-4-carboxy-5,11-bis(carboxymethyl)-1-phenyl-12--
[(phenyl-methoxy)methyl]-8-(phosphonomethyl)-2-oxa-5,8,11-triazatridecan
-13-oic acid;
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxy-
methyl-L-tryptophan];
N,N-Bis[2-[(carboxymethyl)(phosphonomethyl)amino]eth-
yl]-O-(4-hydroxy-phenyl) -3,5-diiodo-L-tyrosine;
N,N'-[(Phosphonomethylimi-
no)di-2,1-ethanediyl]bis[N-(carboxymethyl)-glycine];
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-(phosphonomethyl)-gly-
cine];
N,N'-[[[3-Carboxy-1-phosphonopropyl]imino]di-2,1-ethanediyl]bis[N-(-
carboxymethyl)glycine];
4-Phenyl-N-[trans4-[[[4[bis[2-[bis(carboxymethyl)a-
mino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino]methyl]cyclohexylcarbonyl]--
L-phenylalanina;
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[4-[bis[2-[bis(ca-
rboxymethyl)amino]ethyl]amino]-1-oxo-4phosphonobutyl]amino]-7,12-dihydroxy-
cholan-24-oic acid;
N,N'-[[[3-Amino-1-phosphonopropyl]imino]di-2,1-ethaned-
iyl]bis[N-(carboxymethyl)glycine].
8. Compounds as claimed in any of the claims from 1 to 7 in form of
chelated complexes with metal ions selected from Gd.sup.(3+),
Dy.sup.(3+), Fe.sup.(3+), Fe.sup.(2+) and Mnhu (.sup.2+).
9. Compounds as claimed in claims 1-8 wherein the physiologically
acceptable salifying organic base is selected from: ethanolamine,
diethanolamine, morpholine, glucamine, N,N-dimethylglucamine,
N-methylglucamine, lysine, arginine, ornithine.
10. Compounds as claimed in claim 8 having .tau..sub.m values
<100 ns.
11. Compounds as claimed in claim 10 having .tau..sub.M values
ranging from 10 and 100 ns.
12. Compounds as claimed in claim 11 having .tau..sub.M values
ranging from 20 and 50 ns.
13. A process for the preparation of the compounds of claim 2,
having the formula (II) wherein R.sub.1 is H and R has the meanings
defined above in claim 1, which comprises: a) esterification of the
suitable amino acid; b) N-alkylation of the ester from step a) by
reacting it with a suitable bromoacetate; c) bromoalkylation of the
intermediate from step b) by reacting it with
trifluoromethanesulfonic acid 2-bromoethyl ester prepared from
bromoethanol, trifluoromethanesulfonic anhydride and 2,6-lutidine;
d) preparation of aminomethylphosphonic acid diethyl ester by
direct condensation of tribenzylhexahydrotriazine with a suitable
dialkyl phosphite and subsequent debenzylation by catalytic
hydrogenation of the condensation product; e) bis alkylation of
aminomethylphosphonic acid diethyl ester by reacting it with the
intermediate from step c) and isolation of the hexaester; f)
deprotection of the acidic functions of the hexaester and isolation
of the acid chelating agent.
14. A process as claimed in claim 13 wherein the bromoalkyl
derivative from step c) is alternatively prepared starting from the
corresponding hydroxy derivative by reacting it with a suitable
brominating agent.
15. A process for the preparation of the compounds of claim 2,
having the is formula (II) wherein R is H and R.sub.1 has the
meanings defined above in claim 1, which comprises: a) preparation
of 2,2'-(Iminodi-2,1-ethanedi- yl)bis-1H-isoindole-1,3(2H)-dione)
by reacting phthalic anhydride with diethylenetriamine in acetic
acid; b) N-alkylation of the bis-phthalimido derivative from step
a) by reacting it with 3-benzyloxycarbonylpropionald- ehyde in a
suitable organic medium, and then with tris(tert-butyl)phosphit- e;
c) removal of phthalic groups; d) N-alkylation of the diamine from
step c) by reacting it with a suitable halo acetate; e)
debenzylation by catalytic hydrogenation of intermediate from d)
and isolation of the hexaester monocarboxylic acid derivative; f)
reaction of the hexaester monocarboxylic acid intermediate from e)
with a suitable amino compound and isolation of the corresponding
amide; g) deprotection of the acidic functions of the hexaester and
recovery of the acid chelating agent.
16. A process for the preparation of the compounds of claim 3,
having formula (III), which comprises: a) preparation of
aminomethylphosphonic acid bis tert-butyl ester (bis N-alkyl)
derivative by reacting a suitably activated bis tert-butyl
phosphite with animal and directly transforming the resulting
trimethylsilyl derivative into the corresponding hydroxy derivative
by treatment with a suitable aqueous acid; b) catalytic
hydrogenation of the intermediate from step a); c) N-alkylation of
the resulting compound from step b) by reacting it with a suitable
haloacetate; d) transformation of the aminoalcohol from step c)
into the corresponding bromo derivative by reacting it with
methanesulfonyl chloride and a suitable brominating agent; e)
condensation of the bromo derivative from step d) with a convenient
amino acid suitably esterified and recovery of the polyester; f)
deprotection of the acidic functions of the polyester and recovery
of the chelating agent.
17. A process according to claim 16 wherein in the synthetic step
a) the phosphonic acid tert-butyl ester is activated with
Me.sub.3SiCl.
18. A process according to claim 16 wherein the synthesis of the
aminomethylphosphonic acid bis tert-butyl ester bis N-alkyl
derivative in step a) is catalyzed by a catalytic amount of a
lanthanide triflate.
19. A process according to claim 18 wherein the used lanthanide
triflate is ytterbium triflate.
20. A complex compound according to any one of claims from 1 to 12
for diagnostic use.
21. A contrastographic diagnostic pharmaceutical composition
comprising a chelated complex compound of any one of claims from 1
to 12 in mixture with a suitable carrier.
22. The use of the chelated complex compounds of claims from 1 to
12 for the preparation of diagnostic formulations for the imaging
of organs and/or tissues of the human or animal body, by means of
M.R.I.
Description
[0001] This invention refers to new compounds which can chelate
paramagnetic bi- and trivalent metal ions, their chelates with said
metal ions and their use as contrast agents in magnetic resonance
imaging (M.R.I.).
[0002] From a radiologist's point of view, an improvement in the
radiographic image, which means a better contrast enhancement
between healthy and diseased tissues, is seen as an aid to the
diagnosis which can be obtained through previous administration of
suitable exogenous substances.
[0003] These substances cause a significant alteration of a
specific characteristic, known as relaxivity, of the water protons
belonging to the tissue under examination, when such protons are
submitted to an external magnetic field.
[0004] These substances are known as contrast agents for M.R.I. A
number of chelated complexes of linear and cyclic
polyaminopolycarboxylic ligands with paramagnetic metals are known
to be useful as M.R.I. contrast agents.
[0005] Said compounds generally derive from the two basic
polyaminopolycarboxylic structures, namely
diethylenetriaminopentaacetic acid (DTPA) and
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
(DOTA).
[0006] The compounds of the present invention are novel polyamino
derivatives comprising at least one phosphonic residue as one of
the binding site in the chelating agent structure.
[0007] Relaxivity (r.sub.1p) is an intrinsic property of
paramagnetic complexes which characterizes their ability to
increase the nuclear magnetic relaxation rate of vicinal protons.
In the case of Gd(III) chelates with q.gtoreq.1, wherein q is the
number of coordinated water molecules, a remarkable contribution to
the increase in relaxation observed for water protons of the
solvent derives from the exchange between the molecule(s) of bound
water and the molecules of the remaining solvent (S. Aime et al.,
Chem. Soc. Rev., 1998, 27, 19).
[0008] This contribution (r.sub.1p.sup.is) is related to the
relaxation time (T.sub.1M) and to the residence time (.tau..sub.M)
of the protons of the water molecule(s) which are coordinated in
the inner coordination sphere according to the following equation:
1 r 1 p is = 1.8 10 - 5 q ( T 1 M + M )
[0009] T.sub.1M receives contributions from the reorientation of
the paramagnetic species, .tau..sub.R, through the residence time
of the coordinated water protons, .tau..sub.M, and the electronic
relaxation time of the metal ion, .tau..sub.S. Moreover,
r.sub.1p.sup.is is the highest when T.sub.1M>.tau..sub.M (fast
exchange conditions) and T.sub.1M is as short as possible.
[0010] A remarkable increase in r.sub.1p at the magnetic field
values conventionally used in clinical practice has been up to now
obtained, in different ways, mainly by decreasing molecular
tumbling, with a consequent increase in .tau..sub.R. The expected
increase in r.sub.1p has not, however, been observed due to the
limiting effect caused by the residence time of water molecules,
.tau..sub.1M. A fine tuning of this parameter has become the
primary object of current research in the M.R.I. field, as only
.tau..sub.M values of about 30 ns would make it possible to
completely exploit the decrease in T.sub.1M induced by the increase
in .tau..sub.R. For this reason, the exchange rate values of water
molecules in lanthanide (III) complexes are of paramount importance
in the development of novel M.R.I. contrast agents. In fact, the
residence time of the water molecule(s) coordinated to a Gd(III)
complex plays a particularly important role, in that it directly
contributes to the nucleus-electron dipolar interaction and
controls the transfer efficiency of the paramagnetic effect to the
water molecules of the solvent.
[0011] The above cited prior art contrast agents, generally
comprising polyaminopolycarboxylic acid derivatives, have shown
.tau..sub.M values generally comprised between 200 and 2500 ns,
where such values are significantly higher than the 30 ns optimum
one.
[0012] Optimization and harmonization of the above parameters are
still remarkably important objects for everyone dealing with the
development of novel M.R.I. contrast agents.
[0013] The present invention relates to polyamino derivatives
comprising as the binding site in the structure of the chelating
agent at least one phosphonic residue, capable of causing an
increase in the proton exchange rate and therefore advantageously
low .tau..sub.M values.
[0014] More particularly, the object of the present invention is
acyclic polyamino derivative chelating agents of formula (I), both
in the racemic and the optically active forms, 1
[0015] wherein
[0016] Y is a COOH group or a PO(OH).sub.2 group, with the proviso
that at least one Y group is .dbd.PO(OH).sub.2;
[0017] R is a hydrogen atom, or --(CH.sub.2).sub.m--O--R.sub.2,
(C.sub.1-C.sub.5)-alkyl-(C.sub.6-C.sub.10)-aryl or
(C.sub.1-C.sub.5)-alkyl-heteroaryl whose aryl or heteroaryl moiety
comprises 1 or 2 fused rings optionally substituted with one or
more halogen atoms, OH groups, alkyl(C.sub.1-C.sub.5) groups and/or
an OR.sub.3 group, wherein
[0018] R.sub.2 is (C.sub.1-C.sub.5)-alkyl-(C.sub.6-C.sub.10)-aryl,
optionally substituted with one or more halogen atoms, OH and
(C.sub.1-C.sub.5)-alkyl groups;
[0019] R.sub.3 is (C.sub.6-C.sub.10) aryl optionally substituted
with one or more halogen atoms, OH and/or (C.sub.1-C.sub.5)-alkyl
groups;
[0020] m ranges from 1 to 5;
[0021] R.sub.1 can have the same meanings as R with the proviso
that when Y is PO(OH).sub.2, R.sub.1 is selected from H,
(CH.sub.2).sub.mNH.sub.2, (CH.sub.2).sub.mCOOH or an amido
derivative thereof.
[0022] A further object of the invention are the chelates of said
compounds of formula (1) with the bi- and trivalent ions of metal
elements having atomic number ranging between 20 and 31, 39, 42,
43, 44, 49, or between 57 and 83, as well as the salts thereof with
physiologically compatible organic bases selected from primary,
secondary, tertiary amines or basic amino acids, or with inorganic
bases whose cations selected from sodium, potassium, magnesium,
calcium or mixtures thereof.
[0023] A farther object of the present invention is the use of the
compounds of formula (I), their complexes with paramagnetic metals
and the physiologically compatible salts thereof for the
preparation of pharmaceutical formulations for the imaging of
organs and/or tissues of the human or animal body, by use of
M.R.I.
[0024] Examples Of (C.sub.1-C.sub.5)-alkyl-(C.sub.6-C.sub.10)aryl
groups comprise benzyl, phenethyl, naphthylmethyl wherein the aryl
moiety is optionally substituted with one or more halogen atoms or
OR.sub.3 groups wherein R.sub.3 is as defined above.
[0025] Examples of (C.sub.1-C.sub.5)-alkyl-heteroaryl groups
comprise pyridylmethyl or indolylmethyl.
[0026] Examples Of (C.sub.6-C.sub.10) aryl groups comprise phenyl
or naphthyl optionally substituted with one or more halogen atoms,
OH and/or (C.sub.1-C.sub.5)-alkyl groups.
[0027] Examples Of (C.sub.1-C.sub.5) alkyl groups preferably
comprise methyl, ethyl, isopropyl.
[0028] Preferred are compounds of formula (II), 2
[0029] wherein 4 side carboxylic groups and a central phosphonic
group are present and wherein
[0030] R and R.sub.1 have the above defined meanings. Among
compounds of formula (II), particularly preferred are those in
which R.sub.1 is an hydrogen atom and R can assume all previously
defined meanings.
[0031] Also preferred are the compounds of formula (III) 3
[0032] wherein two side phosphonic groups and three carboxylic
groups are present, and wherein
[0033] R.sub.1 has all the values defined above, as well as the
compounds of general formula (IV), 4
[0034] wherein three phosphonic groups and two carboxylic groups
are present and wherein
[0035] R has the values defined above.
[0036] Particularly preferred are the following compounds:
[0037]
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxymethyl-L-
-phenylalanine];
[0038]
[[4s-(4R*,12R*)]-4Carboxy-5,11-bis(carboxymethyl)-1-phenyl-12-[(phe-
nylmethoxy)methyl]-8-(phosphonomethyl)-2-oxa-5,8,11-triazatridecan-13-oic]-
acid;
[0039]
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxymethyl-L-
-tryptophan];
[0040]
N,N-Bis[2-[(carboxymethyl)(phosphonomethyl)amino]ethyl]-O-(4-hydrox-
yphenyl)-3,5-diiodo-L-tyrosine;
[0041]
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-(carboxymethyl)-
-glycine];
[0042]
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[(N-(phosphonometh-
yl)glycine];
[0043]
N,N'-[[[3-Carboxy-1-phosphonopropyl]imino]di-2,1-ethanediyl]bis[N-(-
carboxymethyl)glycine];
[0044]
4-Phenyl-N-[trans-4-[[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]amin-
o]-1-oxo-4-phosphonobutyl]amino]methyl]cyclohexylcarbonyl]-L-is
phenylalanina;
[0045]
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[4[bis[2-[bis(carboxymethyl-
)amino]ethyl]amino]-1-oxo-4phosphonobutyl]amino]-7,12-dihydroxycholan-24-o-
ic acid;
[0046]
N,N'-[[[3-Amino-1-phosphonopropyl]imino]di-2,1-ethanediyl]bis(N-(ca-
rboxymethyl)glycine];
[0047] as well as the paramagnetic chelated complexes thereof and
the physiologically compatible salts thereof.
[0048] Preferred chelates are those in which the bi- or trivalent
metal ion is selected from Gd(.sup.3+), Dy(.sup.3+), Fe(.sup.3+),
Fe(.sup.2+) and Mn(.sup.2+). Particularly preferred are Gd(.sup.3+)
chelates.
[0049] Preferred cations of inorganic bases optionally suitable for
salifying the chelated complexes of the invention particularly
comprise the ions of alkali or alkaline-earth metals such as
potassium, sodium, calcium, magnesium, and mixtures thereof.
[0050] Preferred cations of organic bases suitable for this purpose
comprise, inter alia, those obtained by protonation of primary,
secondary and tertiary amines such as ethanolamine, diethanolamine,
morpholine, glucamine, N-methylglucamine,
N,N-dimethylglucamine.
[0051] Preferred cations of amino acids comprise, for example,
those of lysine, arginine or ornithine.
[0052] The introduction of at least one phosphonic group as the
binding site in the structure of the chelating agent unexpectedly
provided contrast agents having an advantageous increase in the
proton exchange rate and, therefore, particularly low .tau..sub.M
values.
[0053] In particular, the chelated complexes of the invention are
characterized by .tau..sub.M<100 ns values, preferably values
between 10 and 100 ns, most preferably between 20 and 50 ns.
[0054] Among the various synthetic approaches to the compounds of
the invention, the one preferred for the preparation of the
compounds of formula (II), and particularly for the compounds in
which R.sub.1 is .dbd.H and R has the meanings defined above in
claim 1, is reported in the following Scheme 1: 5
[0055] wherein R has the values defined above for compounds
(I).
[0056] Briefly, the synthetic process of Scheme I comprises the
following steps:
[0057] a) Esterification of a suitable amino acid. Said
esterification can be advantageously carried out by reacting the
amino acid with an alkyl acetate and an acid such as HClO.sub.4. In
a variation of the process, the amino acid, previously N-protected
by reaction with CBZCl, can be esterified by reaction with an alkyl
halide, in the presence of a base such as K.sub.2CO.sub.3;
[0058] b) N-Alkylation of the resulting ester (intermediate 1) by
reacting it with a suitable bromoacetate, such as tert-butyl
bromoacetate. Said reaction is carried out in an organic solvent
preferably selected among acetonitrile, THF, EtOAc and in the
presence of a pH 8 buffer solution;
[0059] c) Bromoalkylation of the intermediate 2 by reacting it with
trifluoromethanesulfonic acid 2-bromoethyl ester (intermediate 3)
previously prepared from bromoethanol, trifluoromethanesulfonic
anhydride and 2,6-lutidine. The bromoalkylation is carried out in
an organic solvent suitably selected among, for example, toluene,
acetonitrile, dichloroethane, and in the presence of an amine
selected among ethylenediamine, diisopropylethylamine,
triethylamine, to give the intermediate 4. In a variation of the
process of the invention, compound 4 can be alternatively prepared
starting from the corresponding N-(2-hydroxyethyl) derivative,
obtained as described in WO 98/05625, (incorporated herein by
reference in its entirety), by reacting it with a brominating agent
such as NBS, in the presence of triphenylphosphine;
[0060] d) Preparation of aminomethylphosphonic acid diethyl ester
(intermediate 5) by direct condensation of
tribenzylhexahydrotriazine with a suitable dialkyl phosphite and
subsequent debenzylation by catalytic hydrogenation of the
condensation product;
[0061] e) Bis alkylation of intermediate 5 by reaction with
intermediate 4 and isolation of hexaester 6. In the process of the
invention, the bis alkylation reaction is preferably carried out in
an organic solvent such as acetonitrile, ethyl acetate, and in the
presence of a pH 8 buffer solution;
[0062] f) Deprotection of the acidic functions of intermediate 6
and isolation of the acid chelating agent 7. Said deprotection can
be obtained by reacting the hexaester with iodotrimethylsilane in
an organic solvent, such as CH.sub.3CN.
[0063] A different synthetic approach for the preparation of the
compounds of formula (II) in which, on the contrary, R is .dbd.H
and R.sub.1 has the meanings defined above in claim 1, is reported
in the following Scheme 1bis: 6
[0064] in which, as an example, is detailed the preparation of one
of several preferred complex compounds of the invention.
[0065] The synthetic process of SCHEME 1bis essentially comprises
the following steps:
[0066] a) Preparation of
2,2'-(Iminodi-2,1-ethanediyl)bis-1H-isoindole-1,3- (2H)-dione)
(intermediate 1) by reacting phthalic anhydride with
diethylenetriamine in acetic acid;
[0067] b) N-alkylation of the bis-phthalimido derivative 1 by
reacting it with 3-benzyloxycarbonylpropionaldehyde (intermediate
2) in a suitable organic medium, and then with tris(tert-butyl)
phosphite to give intermediate 3;
[0068] c) Removal of phthalic groups to give corresponding diamine
(intermediate 4) by reaction, for example, with hydrazine;
[0069] d) N-alkylation of the diamine 4 by reaction with a suitable
halo acetate, such as, for example, tert-butyl bromoacetate, to
give intermediate 5. This reaction is carried out in an organic
solvent preferably selected from acetonitrile, ethyl acetate, and
in the presence of a suitable tertiary amine such as, for example,
diisopropylethylamine;
[0070] e) Debenzylation by catalytic hydrogenation of the
intermediate 5 and isolation of the hexaester monocarboxylic acid
6. In a preferred process this hydrogenation is carried out in an
organic solvent such as, for example, THF and catalysed by 10%
Pd--C;
[0071] f) reaction of the hexaester monocarboxylic acid 6 with a
suitable amino compound (compound 7) and isolation of the
corresponding amide (derivative 8). In a preferred process said
reaction is performed in presence of HATU
(O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluoro-phosphate);
[0072] g) Deprotection of the acidic functions of the hexaester and
recovery of the acid chelating agent (compound 10). In one
preferred process of the invention the deprotection of the acidic
functions is performed on the hexaester derivative prepared at step
e) (intermediate 6) to give, for example, the acid chelating agent
of EXAMPLE 7, disclosed later on in the experimental section of the
invention. In the process of Scheme Ibis, otherwise, the
deprotection includes a first debenzylation step, by catalytic
hydrogenation, of the benzylester contained in the amido derivative
8 and a second step, including the deprotection of the residual
acidic functions of the hexaester 9, to give the chelating agent
10. The hydrogenation is preferentially carried out in an organic
solvent such as, for example, THF and catalysed by 10% Pd--C. The
subsequent deprotection can be performed, for example, by reacting
the hexaester 9 with trifluoroacetic acid.
[0073] On the other hand, compounds of general formula (III) are
preferably prepared according to the following Scheme 2 7
[0074] wherein R.sub.1 has the values defined above for compounds
(I).
[0075] The synthetic process of Scheme 2 comprises the following
steps:
[0076] a) Preparation of aminomethylphosphonic acid bis tert-butyl
ester (bis N-alkyl) derivative (intermediate 3) by reacting bis
tert-butyl phosphite, suitably activated (intermediate 1), with
aminal (intermediate 2). In particular, in the process of the
invention, the phosphonic acid tert-butyl ester is advantageously
activated for example with Me.sub.3SiCl in a reaction carried out
in organic solvent and in the presence of an amine, such as
triethylamine. The resulting trimethylsilyl derivative is reacted
with intermediate 2 obtained from 2-benzylaminoethanol and aqueous
formaldehyde. This reaction is activated by the presence of a
catalytic amount of a lanthanide triflate. Particularly preferred
is ytterbium triflate. In the process of the invention, the
resulting trimethylsilyl derivative is not isolated but it is
directly transformed into the corresponding hydroxy derivative by
treatment with a suitable aqueous acid, such as aqueous acetic
acid.
[0077] b) Catalytic hydrogenation of intermediate 3. In the
preferred process this reaction is carried out in alcoholic medium
and catalyzed by Pd(OH).sub.2/C.
[0078] c) N-alkylation of the resulting compound from step b),
(intermediate 4), by reacting it with a suitable haloacetate, such
as tert-butyl bromoacetate. This reaction is carried out in an
organic solvent preferably selected from acetonitrile, ethyl
acetate, and in the presence of a buffer solution pH 8.
[0079] d) Transformation of the isolated aminoalcohol (intermediate
5) into the corresponding bromo derivative by reacting it with
methanesulfonyl chloride and a brominating agent such as lithium
bromide. This reaction is carried out in an organic solvent
selected from THF, acetonitrile, ethyl acetate, under nitrogen
atmosphere and in the presence of an amine selected from
triethylamine, diisopropylethylamine, at temperatures ranging from
20 to -5.degree. C.
[0080] e) Condensation of the bromo derivative (intermediate 6)
with a convenient amino acid suitably esterified (intermediate 7)
and isolation of the polyester (intermediate 8). Said reaction is
advantageously carried out in an organic solvent preferably
selected from acetonitrile, THF, ethyl acetate and in the presence
of a buffer solution pH 8.
[0081] f) Deprotection of the acidic functions of the polyester and
isolation of the chelating agent (compound 9). The deprotection is,
for example, obtained by reacting the polyester with an acid
selected from HCl, H.sub.2SO.sub.4, in an aqueous mixture of an
organic solvent such as dioxane.
[0082] The compounds of this invention have a wide range of
applications, since they can be used for intravasal, (for instance
i.v., intraarterial, intracoronaric, intraventricular
administration and so on), intrathecal, intraperitoneal,
intralymphatic and intracavital administrations. Furthermore, the
compounds are suitable for the oral or enteral administration, and
therefore, specifically for the imaging of the gastrointestinal
tract.
[0083] For the parenteral administration they can preferably be
formulated as sterile aqueous solutions or suspensions, whose pH
can range from 6.0 to 8.5.
[0084] These aqueous solutions or suspensions can be administered
in concentrations ranging between 0.002 and 1.0 M. These
formulations can be lyophilized and supplied as they are to be
reconstituted before use.
[0085] For the gastrointestinal use or for injection in body
cavities, these agents can be formulated as a solution or
suspension optionally containing suitable excipients in order, for
example, to control viscosity.
[0086] For the oral administration, they can be formulated
according to preparation methods routinely used in the
pharmaceutical technique or as coated formulations to gain
additional protection against the stomach acidic pH, thus
preventing the chelated metal ion from release, which takes place
particularly at the pH values typical of gastric juices.
[0087] Other excipients, such as sweeteners and/or flavouring
agents, can also be added, according to known techniques of
pharmaceutical formulations.
[0088] The solutions or suspensions of the compounds of this
invention can also be fornulated as aerosols to be used in
aerosol-bronchography and instillation.
[0089] The compounds of the present invention can optionally be
chemically conjugated to suitable macromolecules, targeting vectors
or inglobated into suitable carriers.
[0090] For example they can also be encapsulated in liposomes or
they can be constituents of their chemical structure and used as
uni- or multilamellar vesicles.
[0091] A non-limiting list of preferred compounds of the invention
is reported is in the following, to better exemplify the wide
applicative potential of the invention. 8 9 10 11 12 13 14 15 16
17
EXPERIMENTAL SECTION
EXAMPLE 1
[0092] Gadolinium complex of
[N,N'-[(phosphonomethylimino)di-2,1-ethanediy-
l]bis[N-carboxymethyl-L-phenylalanine] salified with Na (1:3)
18
[0093] A) L-phenylalanine 1,1-dimethylethyl ester
[0094] A solution of L-phenylalanine (62.6 g; 379 mol) in
tert-butyl acetate (320 mL) cooled on an ice bath and stirred
vigorously is slowly added with 70% aqueous HClO.sub.4 (35 mL, 407
mol). After stirring for 11 days at room temperature, the mixture
is diluted with 100 mL of water and cooled on an ice bath. The
mixture is basified with 5 N NaOH to precipitate a white solid
(unreacted phenylalanine) which is filtered off. The nixture is
then extracted with EtOAc (4.times.200 mL), the organic phases are
combined and washed with water (2.times.200 mL) and 5%
Na.sub.2CO.sub.3 (300 mL). After drying over Na.sub.2SO.sub.4 and
carefully removing the solvent under vacuum, the desired compound
is obtained as a colourless oil (53.53 g; 242 mol), which needs no
further purifications and is stored at -18.degree. C.
[0095] Yield: 64%
[0096] TLC: Carrier: silica gel plate 60F 254 Merck
[0097] Eluent: CHCl.sub.3/CH.sub.3OH/25% NH.sub.4OH 90:9:1.
[0098] Detection: 0.2% (w/v) ninhydrin in ethanol Rf=0.6
[0099] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0100] B) N-[2-(1,1-Dimethylethoxy)-2-oxoethyl]-L-phenylalanine
1,1-dimethylethyl ester
[0101] An emulsion of L-phenylalanine 1,1-dimethylethyl ester
(compound prepared at point A) (53.53 g; 242 mol), tert-butyl
bromoacetate (37.3 mL; 254 mol) in acetonitrile (400 mL) and 2M
buffer phosphate pH 8 (200 mL) is vigorously stirred at room
temperature for 16 hours. After separation, the organic phase is
evaporated and the residue is taken up into EtOAc; the aqueous
phase is extracted with EtOAc (3.times.200 mL). The combined
organic phases are washed with water (2.times.300 mL), saline
solution (200 mL) and finally dried over Na.sub.2SO.sub.4. The
crude is purified by flash chromatography (n-hexane/EtOAc 9:1 to
75:25). After removing the solvents under vacuum, the desired
compound is obtained as a colourless oil (66.04 g; 196.90 mol).
[0102] Yield: 81%
[0103] TLC: Carrier: silica gel plate 60F 254 Merck
[0104] Eluent: CHCl3/CH.sub.30H 95:5.
[0105] Detection: 0.2% (w/v) ninhydrin in ethanol Rf-0.5
[0106] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0107] C) Trifluoromethanesulfonic acid 2-bromoethyl ester
[0108] 240 g of trifluoromethanesulfonic anhydride (0.805 mol) are
added in 1.5 h, under inert atmosphere, to a solution of
bromoethanol (57 mL; 0.80 mol) and 2,6-lutidine (104 mL; 0.89 mol)
in CH.sub.2Cl.sub.2 cooled at -5.degree. C. After 10 min the
mixture is concentrated to one fourth the volume, then eluted
through a small layer of silica gel (eluent n-hexane/EtOAc=9:1). By
evaporation and drying, the desired product is obtained (147.2 g;
0.57 mol).
[0109] Yield: 72%
[0110] TLC: Carrier: silica gel plate 60F 254 Merck
[0111] Eluent: n-hexane/iPr.sub.2O=8:2
[0112] Detection: 0.5% KMnO.sub.4 in 1M NaOH Rf=0.6
[0113] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0114] D)
N-(2-Bromoethyl)-N-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-phenyla-
lanine 1,1-dimethyl ester
[0115] The intermediate prepared at point C) (147.2 g; 573 mol) is
added under nitrogen to a solution of
N-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-p- henylalanine
1,1-dimethylethyl ester (65.93 g; 197 mol) and 2,6-lutidine (72 mL;
0.62 mol) in 600 mL of dry toluene at -15.degree. C. After 16 h at
room temperature, 200 mL of EtOAc, 200 mL of H.sub.2O and 50 mL of
ethylenediamine are added to the mixture. The organic phase is
washed with is 300 mL of H2O, 100 mL of acetate buffer pH=5.8, 100
mL of saturated aqueous CuSO.sub.4 (hereinafter aqCuSO.sub.4), 200
mL of saturated aqNH.sub.4Cl, dried over Na.sub.2SO.sub.4 and
evaporated. The residue is taken up into iPr.sub.2O and quickly
filtered through a small layer of silica gel. By evaporation of the
filtrate, the desired product is obtained (83.08 g; 188 mol).
[0116] Yield: 95.4%
[0117] GC assay: 97% (in % area)
[0118] TLC: Carrier: silica gel plate 60F 254 Merck
[0119] Eluent: n-hexane/EtOAc=9:1
[0120] Detection: 0.5% KMnO.sub.4 in 1M NaOH Rf=0.5
[0121] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0122] E) [(Phenylmethyl)amino]methylphosphomic acid diethyl
ester
[0123] 1,3,5-Tribenzylhexahydro-1,3,5-triazine (98%; 12.48 g; 34.21
mol) is reacted with diethyl phosphite (94%; 15.5 ml; 113 mol) for
6 hours, under nitrogen atmosphere and at 100.degree. C. The
mixture is then cooled to room temperature, taken up with ethyl
ether (150 mL) and acidified with 6N HCl (20 mL). The organic phase
is extracted with 1N HCl (10 mL), the aqueous phases are basified
with 5N KOH, then extracted with Et.sub.2O (300+150 mL), washed
with brine (100 mL) and finally dried over Na.sub.2SO.sub.4. After
evaporation of the solvent under vacuum, the product is recovered
as a colourless oil (25.05 g; 97.37 mol) which is stored at a
temperature of -18.degree. C.
[0124] Yield: 95%
[0125] TLC: Carrier: silica gel plate 60F 254 Merck
[0126] Eluent: toluene/EtOAc/iPrOH 7:2:1
[0127] Detection: 254 nm; 0.5% KMnO.sub.4 in 1M NaOH Rf=0.38
[0128] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0129] F) Aminomethylphosphonic acid diethyl ester
[0130] A solution of the compound prepared at point E (28.3 g; 110
mol) in methanol (600 mL) is stirred vigorously in the presence of
Pd(OH).sub.2/C (32 g) and HCOONH.sub.4 (120 g) for 6 hours. The
mixture is then filtered through a layer of Celite.RTM. and the
filtered solution is evaporated to give a residue, which is taken
up in ethanol (200 mL) and treated with Amberlite.RTM. IRA 400
resin in the OH-- form (150 mL, previously conditioned with
absolute ethanol) for 3 hours. The suspension is then filtered and
the solution is evaporated to dryness to obtain an oil (20.47 g)
which is purified by flash chromatography
(CH.sub.2Cl.sub.2/CH.sub.3OH/25% NH4OH 954:40:6 to 89:10:1) to
obtain 15.09 g (90.3 mol) of product as a colourless oil.
[0131] Yield: 82%
[0132] TLC: Carrier: silica gel plate 60F 254 Merck
[0133] Eluent: CH.sub.2Cl.sub.2/CH.sub.3OH/NH.sub.4OH 25%
89:10:1
[0134] Detection: 254 nm; 0.5% KMnO.sub.4 in 1M NaOH; 0.2% (w/v)
ninhydrin in ethanol RF=0.5
[0135] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0136] G)
N,N'-[[[(Diethoxyphosphinyl)methyl]imino]di-2,1-ethanediyl]bis[N-
-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-phenylalanine]1,1-dimethylethyl
ester
[0137] An emulsion of the aminophosphonate prepared at point F)
(8.29 g; 49.6 mol), bromide (prepared at point D) (62.92 g; 103.8
mol) in CH.sub.3CN (300 mL) and 2M phosphate buffer pH=8 (200 mL)
is stirred vigorously at room temperature for 16 h. After replacing
the aqueous phase with fresh buffer (200 mL) the mixture is stirred
for a further 32 h. The organic phase is evaporated under reduced
pressure, taken up with EtOAc, and the aqueous phase is repeatedly
extracted with EtOAc (3.times.150 mL). The combined organic phases
are washed with H.sub.2O, brine, dried over Na.sub.2SO.sub.4 and
evaporated under reduced pressure. The crude product is purified by
flash chromatography (eluent n-hexane/EtOAc/iPrOH=7:3:0.1 to
6:4:0.2) to give the desired product. (31.38 g; 35.25 mol).
[0138] Yield: 71%
[0139] HPLC assay: 97% (in % area)
[0140] TLC: Carrier: silica gel plate 60F 254 Merck
[0141] Eluent: n-hexane/iPr.sub.2O=65:35
[0142] Detection: 0.5% KMnO.sub.4 in 1M NaOH Rf=0.6
[0143] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0144] H)
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxymethy-
l-L-phenylalanine]
[0145] To a solution of the hexaester prepared at point G) (31.21
g; 35.06 mol) in 500 mL of CH.sub.3CN is slowly added, at
-15.degree. C, under inert atmosphere, iodotrimethylsilane (80 mL;
588 mol). The mixture is then stirred at room temperature for 3
days. After cooling on ice and adding H20 (300 mL), volatiles are
removed under reduced pressure and the pH of the residual mixture
is adjusted to 8 with 6N NaOH. The crude is then purified by
chromatography through Amberlite.RTM. XAD 1600 resin eluting with
water, 7% aq. Na.sub.2SO.sub.3 and finally with a
H.sub.2O/CH.sub.3CN gradient (95:5 30:70) to afford the desired
product (19.66 g; 32.25 mol).
[0146] Yield: 92%
[0147] HPLC assay: 100% (in % area)
[0148] Elemental analysis
1 C H N P % calc.: 53.20 5.95 6.89 5.08 % found: 52.93 6.18 6.83
4.21 H.sub.2O 1.51%
[0149] TLC: Carrier: silica gel plate 60F 254 Merck
[0150] Eluent: n-hexane/EtOAc=9:1
[0151] Detection: 0.5% KMnO.sub.4 in 1M NaOH Rf=0.5
[0152] Specific rotation: [.alpha.].sub.589.sup.20=+8.4;
[.alpha.].sub.578.sup.20=+8.7; [.alpha.].sub.546.sup.20=+10.6;
[.alpha.].sub.436.sup.20=+24.9; [.alpha.].sub.405.sup.20=+34.1;
[.alpha.].sub.365 .sup.20=+55.1 (c 1.17; 0.5NNaOH)
[0153] .sup.1H-NMR, .sup.13C-NMR, .sup.31P-NMR, IR and MS spectra
are consistent with the indicated structure.
[0154] 1) Gadolinium complex of
N,N'-[(phosphonomethylimino)di-2,1-ethaned-
iyl]bis[N-carboxymethyl-L-phenylalanine]trisodium salt
[0155] To an aqueous solution of the compound prepared at point H
(19.66 g, 32.25 mol) is added GdCl.sub.3.6H.sub.2O (32.25 mol) and
2N NaOH keeping pH within the range 6-7. The progress of the
reaction is checked by HPLC. After 18 h the solution is filtered
through a Millipore.RTM. filter, nanofiltered and concentrated (250
mL). The desalted solution is slowly percolated through a
Dowex.RTM. CCR3LB column (Na.sup.+ form; 35 mL) to obtain the
desired product (25.48 g; 30.71 mol).
[0156] Yield: 95%
[0157] m.p.: >210.degree. C. (dec.)
[0158] HPLC assay: 100% (in % area)
[0159] Elemental analysis:
2 C H N Gd Na P % calc.: 39.08 3.64 5.06 18.95 8.31 3.73 % found:
37.61 4.13 5.14 17.03 7.96 3.40 H.sub.2O 9.47%
[0160] Specific rotation: [.alpha.].sub.589.sup.20-35.4;
[.alpha.].sub.578.sup.20=-36.8; [.alpha.].sub.546.sup.20=-42.5;
[.alpha.].sub.436.sup.20=-70.6; [.alpha.].sub.405.sup.20=-84.6;
[.alpha.].sub.365.sup.20=-110,0 (c 1.40; H.sub.2O)
[0161] IR and MS spectra are consistent with the indicated
structure.
EXAMPLE 2
[0162] Gadolinium complex of
[4S-(4R*,12R*)]-4-Carboxy-5,11-bis(carboxymet-
hyl)-1-phenyl-12-[(phenylmethoxy)methyl]-8-(phosphono
methyl)-2-oxa-5,8,11-triazatridecan-13-oic acid, salified with Na
(1:3) 19
[0163] A)
N-(2-Bromoethyl)-N-[2-(1,1-dimethylethoxy)-2-oxoethyl]-O-(phenyl-
methyl)-L-serine 1,1-dimethylethyl ester
[0164] This intermediate is prepared analogously to that of Example
1, following the synthetic steps summarized in Scheme 1.
[0165] Alternatively, in a variation of the present process and in
particular in this case, intermediate 4 is prepared as follows:
[0166] to a solution of
N-[2-(1,1-dimethylethoxy)-2-oxoethyl]-N-(2-hydroxy-
ethyl)-O-(phenylmethyl)-L-serine 1,1-dimethylethyl ester (prepared
as described in WO 98/05625) (61.7 g; 150.7 mol) and triethylamine
(31 mL; 0.22 mol) in dry THF (600 mL) are slowly added, under
nitrogen atmosphere, methanesulfonyl chloride (12.5 mL; 160 mol)
and lithium bromide (111 g; 1.25 mol) at -15/-10.degree. C.
[0167] After evaporating the solvent and dissolving the residue in
toluene and diethyl ether, the solution is washed with water and
brine, then dried over Na.sub.2SO.sub.4 and evaporated to dryness,
to obtain the title product as a colourless oil (69.79 g; 147.7
mol).
[0168] Yield: 98%
[0169] TLC: Carrier: silica gel plate 60F 254 Merck
[0170] Eluent: n-hexane/tOAc=8:2
[0171] Detection: 0.5% KMnO.sub.4 in 1M NaOH; I.sub.2; 254 nm;
Rf=0.75
[0172] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0173] B)
[4S-(4R*,12R*)]-8-[(Diethoxyphosphinyl)methyl]-4-[(1,1-dimethyle-
thoxy)carbonyl]-5,11-bis[2-(1,1-dimethylethoxy)-2-oxoethyl]-1-phenyl-12-[(-
phenylmethoxy)methyl]-2-oxa-5,8,11-triazatridecan-13-oic acid
1,1-dimethylethyl ester
[0174] An emulsion of the aminophosphonate prepared as described in
1F (11.23g; 67.20 mol) and the bromide, prepared as described in
2A, (68.72 g; 145.5 mol) in CH.sub.3CN (250 mL) and 2M phosphate
buffer pH=8 (200 mL) is stirred vigorously at room temperature for
24 h. The phases are separated, the aqueous phase is repeatedly
extracted with EtOAc (250+100 mL) and the organic phase is
evaporated under reduced pressure, then taken up with EtOAc. The
combined organic phases are washed with H.sub.2O, brine and dried
over Na.sub.2SO.sub.4. The crude is purified by flash
chromatography (first column: eluent toluene/EtOAc/iPrOH=80:18:2 to
66:31:3; second and third columns: eluent n-hexane/EtOAc/iPrOH
66:32:2). The solvent is evaporated under vacuum to obtain the
product as a colourless oil (44.84 g; 47.19 mol).
[0175] Yield: 70%
[0176] TLC: Carrier: silica gel plate 60F 254 Merck
[0177] Eluent: n-hexane/EtOAc/iPrOH=50:45:5.
[0178] Detection: 0.5% KMnO.sub.4 in 1M NaOH; I.sub.2; 254 nm;
Rf=0.5
[0179] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0180] C) [4S-(4R*,
12R*)]-4-Carboxy-5,11-bis(carboxymethyl)-1-phenyl-12-[-
(phenylmethoxy)methyl]-8-(phosphonomethyl)-2-oxa-5,8,11-triazatridecan-13--
oic acid
[0181] To a solution of the hexaester prepared at point B (42.03 g;
44.23 mol) in 600 mL of CH.sub.3CN is slowly added at -15.degree.
C., under inert atmosphere, iodotrimethylsilane (80 mL; 588 mol).
The mixture is stirred at room temperature for 24 hours. After
cooling on ice and adding water (150 mL), volatiles are removed
under reduced pressure and pH is adjusted to 8 with 6N KOH. The
resulting solution is concentrated, washed with Et.sub.2O/EtOAc 1:1
(2.times.250 mL), CH.sub.2Cl.sub.2 (2.times.250 mL), heated to
50.degree. C. and acidified to pH 2.6 with 6N HCl. After addition
of CH.sub.3CN (100 mL), the still hot resulting mixture is slowly
loaded onto a column of Amberlite.RTM. XAD 1600 resin which is then
eluted with water and subsequently with a H.sub.2O/CH.sub.3CN
gradient (95:5 to 50:50), until complete elution of the product.
The eluent mixture should be periodically heated to avoid
precipitation of the product on to the column.
[0182] After evaporation, the desired product is obtained as a
white solid (23.52 g; 35.12 mol).
[0183] Yield: 79%
[0184] HPLC assay: 100% (in % area)
[0185] Elemental analysis
3 C H N P % calc.: 52.02 6.02 6.28 4.63 % found: 51.07 6.16 6.31
4.35 H.sub.2O 2.67%
[0186] Specific rotation: [.alpha.].sub.589.sup.20+14.5;
[.alpha.].sub.578.sup.20=+14.9; [.alpha.].sub.546.sup.20=+17.0;
[.alpha.].sub.436.sup.20=+29.0; [.alpha.].sub.495.sup.20=+35.4;
[.alpha.].sub.365.sup.20=+46.5 (c 1.06; CH.sub.3OH)
[0187] .sup.1H-NMR, .sup.13C-NMR, .sup.31P-NMR, IR and MS spectra
are consistent with the indicated structure.
[0188] D) Gadolinium complex of [4S-(4R*,
12R*)]-4-Carboxy-5,11-bis(carbox-
ymethyl)-1-phenyl-12-[(phenylmethoxy)methyl]-8-(phosphonomethyl)-2-oxa-5,8-
,11-triazatridecan-13-oic acid, salified with Na (1:3)
[0189] To a solution of the ligand prepared at point C (22.14 g;
33.06 mol) in a H.sub.2O/CH.sub.3CN 8:1 mixture (0.5 L) is added
Gd.sub.2O.sub.3 (5.936 g; 16,37 mol) and 1N NaOH (80 mL). The
reaction mixture is kept at 60.degree. C. for 20 hours following
the progress of the reaction by HPLC. The suspension is then
filtered through a Millipore.RTM. filter, concentrated (120 mL) and
percolated onto a Dowex.RTM. CCR3LB column (Na.sup.+ form; 50 mL).
The eluate is first treated with Carbopuron.RTM. 2S, then filtered
through paper and through Millipore.RTM. VC 0.1 filter pm. After
evaporation the product is obtained as a white solid (30.51 g;
34.29 mol).
[0190] Yield: about 100%
[0191] m.p.: >250.degree. C. (dec.)
[0192] HPLC assay: 100% (in % area)
[0193] Elemental analysis:
4 C H N Gd Na P % calc.: 39.15 3.85 4.72 17.67 7.75 3.48 % found:
35.62 4.46 4.43 16.00 6.86 2.86 H.sub.2O 8.54%
[0194] Specific rotation: [.alpha.].sub.589.sup.20=-26.0;
[.alpha.].sub.578.sup.20=-27.4; [.alpha.].sub.546.sup.20=-31.2;
[.alpha.].sub.436.sup.20=-50.0; [.alpha.].sub.405.sup.20=-58.3;
[.alpha.].sub.365.sup.20=-72.1 (c 1.115; H.sub.2O)
[0195] IR and MS spectra are consistent with the indicated
structure.
EXAMPLE 3
[0196] Gadolinium complex of
N,N'-[(phosphonomethylimino)di-2,1-ethanediyl-
]bis[N-carboxymethyl-L-typtophan], salified with Na (1:3) 20
[0197] A) N-[(Phenylmethoxy)carbonyl]-L-tryptophan
1,1-dimethylethyl ester
[0198] To a suspension of N-[(phenylnethoxy)carbonyl]-L-tryptophan
(33.27 g; 98.32 mol) (previously prepared by reacting L-tryptophan
with CBZCl, in H.sub.2O and 1N NaOH), benzyltriethylammonium
chloride (BTEAC) (22.4 g; 98.32 mol) and K.sub.2CO.sub.3 (176.91 g;
1.28 mol) in dimethylacetamide (750 mL) is added tert-butyl bromide
(265 mL; 2.36 mol). The solution is heated to 55.degree. C. and
vigorously stirred for 19 hours. The mixture is then cooled to room
temperature, diluted with H.sub.2O (3L) and then extracted with
EtOAc (2.times.1 L). The organic phases are evaporated to dryness
to obtain the title product as a pale yellow oil (36 g; 98 mol)
[0199] Yield: 99%
[0200] HPLC assay: 99% (in % area)
[0201] TLC: Carrier: silica gel plate 60F 254 Merck
[0202] Eluent: n-hexane/EtOAc=7:3
[0203] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 254 nm; Rf=0.44
[0204] .sup.1H-NMR .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0205] B) N-[2-(1,1-Dimethylethoxy)-2-oxoethyl]-L-tryptophan
1,1-dimethylethyl ester
[0206] To a solution of L-tryptophan 1,1-dimethylethyl ester (17.10
g; 65.68 mol), obtained by catalytic hydrogenation of the product
prepared at point A, in CH.sub.3CN (150 mL) and 2M buffer phosphate
(pH 8; 150 mL) is added tertbutyl bromoacetate (10.7 mL; 72.25 mol)
and the resulting mixture is left for 23 hours under strong
stirring. The organic phase is separated and concentrated to a
residue which is purified by flash chromatography (eluent
n-hexane/EtOAc, 8:2) to afford the desired product as pale red oil
(20.07 g; 53.59 mol).
[0207] Overall yield: 54.5% (from L-tryptophan)
[0208] HPLC assay: 100% (in % area)
[0209] TLC: Carrier: silica gel plate 60F 254 Merck
[0210] Eluent: n-hexane/EtOAc=7:3
[0211] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 254 nm; Rf=0.28
[0212] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0213] C)
N-[2-(1,1-Dimethylethoxy)-2-oxoethyl]-N-(2-hydroxyethyl)-L-trypt-
ophan 1,1-dimethylethyl ester
[0214] A jacketed reactor is loaded with a solution of
N-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-tryptophan
1,1-dimethylethyl ester (5 g; 13;35 mol) prepared at point B, in
CH.sub.3CN (25 mL). To the solution cooled to -80.degree. C. is
added ethylene oxide (13 mL; 0.26 mol) and ytterbium triflate (0.83
g; 1.34 mol). The mixture is then slowly warmed to room temperature
then, after 15 hours, diluted with water (50 mL) and extracted with
Et.sub.2O (3.times.50 mL). The organic phases are evaporated to
dryness to obtain a crude which is purified by flash chromatography
(eluent n-hexane/EtOAc 7:3) to give the desired product as a pale
yellow oil (4.32 g; 10.32 mol).
[0215] Yield: 77%
[0216] HPLC assay: 99% (in % area)
[0217] TLC: Carrier: silica gel plate 60F 254 Merck
[0218] Eluent: n-hexane/EtOAc=7:3
[0219] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 254 nm; Rf=0.23
[0220] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0221] D)
N-(2-Bromoethyl)-N-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-tryptop-
han 1,1-dimethylethyl ester
[0222] To a solution of intermediate C (4.64 g; 11.09 mol) in
CH.sub.2Cl.sub.2 (44 mL) is added, under nitrogen, Ph.sub.3P (2.9
g; 11.09 mol). To the resulting mixture cooled to 0.degree. C. is
added NBS (1.97 g; 11.09 mol) in portions. After 3 hours at
0.degree. C. and 1 hour at room temperature, the solution is
concentrated until Ph.sub.3PO precipitates as a white solid. The
mixture is then kept at 4.degree. C. for 72 hours to complete
precipitation. The precipitate is filtered and the filtrate is
concentrated to give a crude which is purified by flash
chromatography (eluent n-hexane/EtOAc 8:2) to afford the desired
product as a pale yellow oil (4.46 g; 9.26 mol).
[0223] Yield: 83%
[0224] HPLC assay: 94% (in % area)
[0225] TLC: Carrier: silica gel plate 60F 254 Merck
[0226] Eluent: n-hexane/EtOAc=8:2
[0227] Detection: 0.5% KMuO.sub.4 in 1M NaOH; 254 nm; Rf=0.42
[0228] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0229] E)
N,N'-[[[(Diethoxyphosphinyl)methyl]imino]di-2,1-ethanediyl]bis[N-
-[2-(1,1-dimethylethoxy)-2-oxoethyl]-L-tryptophan 1,1-dimethylethyl
ester]
[0230] To a solution of aminomethylphosphonic acid diethyl ester
(prepared as in example 1F) (5.25 g; 31.41 mol) and of the
intermediate prepared at point 3D (30.25 g; 62.82 mol) in
CH.sub.3CN (100 mL) is added 2M phosphate buffer pH 8 (200 mL). The
resulting biphasic mixture is kept under strong stirring for 18
hours; the organic layer is then separated and concentrated to
obtain a crude oil which is purified by flash chromatography
(eluent n-hexane/EtOAc/CH.sub.3OH 9:1:0.5) to give the hexaester as
a waxy solid (21.6 g; 22.3 mol).
[0231] Yield: 71%
[0232] HPLC assay: 100% (in % area)
[0233] TLC: Carrier: silica gel plate 60F 254 Merck
[0234] Eluent: n-hexane/EtOAc/CH.sub.3OH=9:1:0.5
[0235] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 254 1m; Rf=0.40
[0236] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the is indicated structure.
[0237] F)
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxymethy-
l-L-tryptophan]
[0238] To a solution of the hexaester (15.17 g; 15.67 mol) prepared
as in point E, in CH.sub.3CN, (200 mL) is added, under nitrogen
atmosphere and at 0.degree. C., (CH.sub.3).sub.3SiI (32 mL; 0.235
mol). The solution is then stirred at room temperature for 22
hours. After cooling to -5.degree. C., the mixture is diluted with
H.sub.2O (30 mL) and washed with Et.sub.2O (2.times.400 mL). The
aqueous layer is separated, neutralized to pH 7 with 2N NaOH and
concentrated to 80 mL. After cooling to 0-5.degree. C., the mixture
is acidified by addition of 2N HCl (32 mL) to obtain a precipitate
which is filtered, washed with H.sub.2O and dried to obtain the
desired product as a crystalline white solid (8.37 g, 12.17
mol).
[0239] Yield: 78%
[0240] m.p.: 157-160.degree. C.
[0241] HPLC assay 98% (in % area)
[0242] Elemental analysis
5 C H N P % calc.: 54.15 5.57 10.18 4.50 % found.: 50.13 5.80 9.34
4.35 H.sub.2O 7.78%
[0243] Specific rotation: [.alpha.].sub.589.sup.20=-10.69;
[.alpha.].sub.578.sup.2=-11.19; [.alpha.].sub.546.sup.20=-12.38;
[.alpha.].sub.436.sup.2016.54; (c 2.02; NaOH 0.1N)
[0244] .sup.1H-NMR, .sup.13C-NMR, .sup.31P-NMR, IR and MS spectra
are consistent with the indicated structure.
[0245] G) Gadolinium complex of
N,N'-[(phosphonomethylimino)di-2,1-ethaned-
iyl]bis[N-carboxymethyl-L-tryptophan]trisodium salt
[0246] To a suspension of chelating agent (6.0 g; 8.72 mol)
prepared as in point F in H.sub.2O (80 mL) cooled to 5.degree. C.
is added at first 1N NaOH (28 mL) to obtain a clear solution, and
then GdCl.sub.3 (0.17M solution) (51 mL; 8.72 mol) keeping pH at 7
by simultaneous addition of 1N NaOH. The solution is left at room
temperature for 1 hour, filtered through Millipore.RTM. HAWP 0.45
filter .mu.m and subsequently percolated through an Amberlite.RTM.
XAD 1600 column eluting with H.sub.2O. The eluate is evaporated to
dryness to obtain the product as a white solid (7.12 g; 7.85
mol).
[0247] Yield: 90%
[0248] m.p.: >250.degree. C. (dec.)
[0249] HPLC assay. 99.6% (in % area)
[0250] Elemental analysis:
6 C H N Gd Na P % calc.: 41.02 3.55 7.71 17.32 7.60 3.41 % found.:
36.18 4.42 7.55 16.79 6.54 3.18 H.sub.2O 11.74%
[0251] Specific rotation: [.alpha.].sub.589.sup.20=-20.96;
[.alpha.].sub.578.sup.20=-21.60; [.alpha.].sub.546.sup.20=-24.55;
[.alpha.].sub.436.sup.20=-40.61; [.alpha.].sub.405.sup.20=-47.83;
[.alpha.].sub.365.sup.20=-60.85 (c 2,505; H2O)
[0252] IR and MS spectra are consistent with the indicated
structure.
EXAMPLE 4
[0253] Gadolinium complex of
N,N-Bis[2-[(carboxymethyl)(phosphonomethyl)am-
ino]ethyl]-O-(4hydroxyphenyl)-3,5-diiodo-L-tyrosine salified with
sodium (1:5) 21
[0254] A) [[(2-Hydroxyethyl)(phenylmethyl)amino]methyl]phosphonic
acid 1,1-dimethylethyl ester
[0255] To a solution of 2-beuzylaminoethanol (30.59 g; 196 mol) in
water (30 mL), cooled on an ice bath, is added formaldehyde (35%
aqueous solution, 16.3 mL; 205 mol). After 5 minutes the mixture is
warmed to room temperature then extracted with CHCl.sub.3
(3.times.40 mL). The organic phases are dried over MgSO.sub.4,
evaporated to dryness recovering the animal (intermediate 2 of
Scheme 2) as a colourless oil which is further dried over
P.sub.2O.sub.5, under vacuum.
[0256] To a solution of di-tert-butyl phosphite (38.12 g; 196 mol)
and triethylamine (28.0 mL; 200 mol) in CH.sub.2Cl.sub.2 (300 mL)
is slowly added (in 30 min) chlorotrimethylsilane (26.5 mL; 197
mol) and stirred for 10 min. To the resulting mixture, containing
the intermediate, is then added a solution of the above prepared
animal in CH.sub.2Cl.sub.2 (100 mL), then ytterbium triflate (12.36
g; 19.9 mol) and the reaction is kept at room temperature for 1.5
hours. After addition of water and further CH.sub.2Cl.sub.2,
insoluble salts are filtered through Celite.RTM. and the solution
is concentrated under vacuum. The resulting intermediate is not
isolated, but is diluted with an AcOH/THF/H2O mixture 3:1:1 (250
mL) and the resulting homogeneous solution is concentrated under
vacuum; most AcOH is removed by azeotropic distillation with
toluene and H.sub.2O and the pH of the solution is adjusted to
neutrality by addition of Na.sub.2CO.sub.3. The mixture is
extracted with EtOAc (3.times.80 mL), the organic phase is dried
over MgSO.sub.4 and then evaporated to dryness to obtain the
product as a colourless oil (43.46 g; 122 mol).
[0257] Yield: 62%
[0258] TLC: Carrier: silica gel plate 60F 254 Merck
[0259] Eluent: iPr.sub.2O/CH.sub.2Cl.sub.2/iPrOH=70:25:5
[0260] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 254 nm; 12;
Rf=0.38
[0261] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0262] B) [[(2-Hydroxyethyl)amino]methyl]phosphonic acid
1,1-dimethylethyl ester
[0263] To a solution of intermediate A (43.46 g; 122 mol) in dry
MeOH (1 L) is carefully added, under nitrogen, Pd(OH).sub.2/C (40
g) as a catalyst. The mixture is then vigorously stirred in an
H.sub.2 atmosphere and for 2 h. After filtration through MgSO.sub.4
and Celitea the resulting solution is evaporated. Residual methanol
is evaporated by azeotropic distillation with cyclohexane to obtain
the desired intermediate as a colourless oil (39.45 g) which,
although containing traces of solvents, can be used (as it is) in
the subsequent reaction without further purification.
[0264] TLC: Carrier: silica gel plate 60F 254 Merck
[0265] Eluent: CH.sub.2Cl.sub.2/CH.sub.3CH/25% (w/w) NH.sub.4OH
89:10:1
[0266] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 2% ninhydrin in
ethanol; Rf=0.5
[0267] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0268] C)
N-[[Bis(1,1-dimethylethoxy)]phosphonomethyl]-N-(2-hydroxyethyl)--
glycine 1,1-dimethylethyl ester
[0269] An emulsion of intermediate B as it is isolated (39.45 g),
tert-butyl bromoacetate (17.8 mL; 121 mol) in acetonitrile (250 mL)
and 2M buffer phosphate pH 8 (200 mL) is vigorously stirred at room
temperature for 4 days. The phases are separated; the organic phase
is evaporated and the residue is treated with EtOAc (3.times.150
mL). The combined organic phases are washed with H.sub.2O
(2.times.200 mL), brine (100 mL) and dried over Na.sub.2SO.sub.4.
The crude is purified by flash chromatography eluting at first with
iPr.sub.2O/CH.sub.2Cl.sub.2/iPrOH=70- :30:2, then with
Et.sub.2O/CH.sub.2Cl.sub.2/iPrOH 70:30:2 to 60:40:3 to afford the
desired intermediate as a colourless oil (28.71 g; 75.27 mol)
[0270] Yield: 62%
[0271] TLC: Carrier: silica gel plate 60F 254 Merck
[0272] Eluent: iPr.sub.2O/CH.sub.2Cl.sub.2/iPrOH=60:35:5
[0273] Detection: 0.5% KMnO.sub.4 in 1M NaOH; I.sub.2; Rf=0.4
[0274] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0275] D)
N-[[Bis(1,1-dimethylethoxy)]phosphonomethyl]-N-(2-bromoethyl)gly-
cine 1,1-dimethylethyl ester
[0276] To a solution of intermediate C and triethylamine in dry THF
(500 mL) cooled at -15.degree. C., is slowly added, under nitrogen,
methanesulfonyl chloride (2.8 mL; 36.1 mol). After 1.5 hours at
-10.degree. C. lithium bromide (25.0 g; 288 mol) is added and the
mixture is left under strong stirring for 16 hours while gradually
warming to room temperature. Most volatiles are then evaporated off
under reduced pressure; the residue is diluted with EtOAc (300 mL)
and Et2O (300 mL) and washed with H.sub.2O (2.times.200 mL). The
organic phase is washed with H.sub.2O/brine 1:1 (200 mL), brine
(100 mL) and finally dried over Na.sub.2SO.sub.4. After evaporation
of the solvents the crude residue is purified by flash
chromatography (eluent n-hexane/Et.sub.2O/iPrOH 1:1:0.01) to afford
the desired intermediate (12.35 g; 27.79 mol) which crystallizes on
storing at -18.degree. C.
[0277] Yield: 83%
[0278] m.p.: 50-51.degree. C.
[0279] TLC: Carrier: silica gel plate 60F 254 Merck
[0280] Eluent: iPr.sub.2O/EtOAc=8:2
[0281] Detection: 0.5% KMnO.sub.4 in 1M NaOH; I.sub.2; Rf=0.35
[0282] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0283] E)
N,N-Bis[2-[[2-(1,1-dimethylethoxy)-2-oxoethyl][[(1,1-dimethyleth-
oxy)phosphinyl]methyl]amino]ethyl]-O-(4-hydroxyphenyl)-3,5-diiodo-L-tyrosi-
ne 1,1-dimethylethyl ester
[0284] An emulsion of intermediate D (12.05 g; 27.12 mol) and
3,5-diiodo tyrosine methyl ester (5.85 g; 10.85 mol) in
acetonitrile and 2M phosphate buffer pH 8 is vigorously stirred at
room temperature for 72 hours. The aqueous phase is replaced with
fresh buffer and the solution is left under stirring for a further
three days. The phases are then separated; the aqueous layer is
extracted with EtOAc (250+100 mL), the organic layer is evaporated
and the residue is taken up into EtOAc. The combined EtOAc
solutions are washed with H.sub.2O (100 mL), H.sub.2/O brine 1:1
(100 mL), brine (100 mL). After drying over Na.sub.2SO.sub.4 and
evaporation of the solvent the residue is purified by flash
chromatography (eluent n-hexane/Et.sub.2O/iPrOH 72:20:8). The
desired intermediate is obtained (intermediate 8, according to
scheme 2) as a colourless oil (11.32 g; 8.94 mol).
[0285] Yield: 82%
[0286] HPLC assay: 98.2% (in % area)
[0287] TLC: Carrier: silica gel plate 60F 254 Merck
[0288] Eluent: toluene/EtOAc/iPrOH=1:1:0.02
[0289] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 254 no; I.sub.2;
Rf=40
[0290] .sup.1HNMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0291] F)
N,N-Bis[2-[(carboxymethyl)(phosphonomethyl)amino]ethyl]-O-(4-hyd-
roxyphenyl)-3,5-diiodo-L-tyrosine
[0292] To a solution of polyester E) (24.9 g; 19.6 mol) in
1,4-dioxane (100 mL) is added 4N HCl (180 mL). The solution is
heated to 70.degree. C. for 3 hours then at 90.degree. C. for 1
hour, following the progress of the reaction by HPLC. The mixture
is cooled to room temperature, concentrated to 200 mL and slowly
loaded onto an Amberlite.RTM. XAD 1600 column. After a first
elution with H.sub.2O, an elution gradient based on
H.sub.2O/CH.sub.3CN is used to afford the desired product as a
white solid (15.5 g; 16.86 mol).
[0293] Yield: 86%
[0294] HPLC assay: 100% (in % area)
[0295] Elemental analysis
7 C H N I P % calc.: 32.81 3.63 4.59 27.23 6.77 % found: 31.11 3.78
4.33 26.11 6.57 H.sub.2O 3.96%
[0296] Specific rotation: [.alpha.].sub.589.sup.20=+7.5;
[.alpha.].sub.578.sup.20=+7.8; [.alpha.].sub.546.sup.20=+9.2;
[.alpha.].sub.436.sup.20=+23.1; [.alpha.].sub.365 .sup.20=+33.3 (c
1.03; CH.sub.3COOH /HCl 6N 4:1)
[0297] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0298] G) Gadolinium complex of
N,N-Bis[2-[(carboxymethyl)(phosphonomethyl-
)amino]ethyl]-O-(4-hydroxyphenyl)-3,5-diiodo-L-tyrosine salified
with sodium (1:5)
[0299] To an aqueous solution of ligand F) (15.33 mol) at pH 7 is
added Gd.sub.2O.sub.3 (2.80 g; 15.39 mol). The solution is diluted
to 2 L with H.sub.2O and heated at 70.degree. C. for 6 hours
following the progress of the complexation by is HPLC. Since the
conversion is only about 14.5%, the solution is added, under
stirring, with a solution of 0.164 M aqGdCl.sub.3 (78.0 mL; 12.8
mol), keeping pH at about 7 with 2N NaOH. After completion of the
complexation the mixture is filtered through a Millipore.RTM. HAWP
0.45 .mu.m filter, concentrated to 1 L and percolated through a
Dowex.RTM. CCR3LB column, Na.sup.+ form. The eluate is concentrated
to 250 mL and nanofiltered. After concentration under vacuum, the
dark solution obtained is treated with Carbopuron.RTM. 2S at
60.degree. C., filtered and finally freeze-dried to obtain a solid
which still contains chloride ions, which is then dissolved in
H.sub.2O and purified by elution over Amberlite.RTM. XAD 1600,
eluting at first with H.sub.2O, then with a H.sub.2O/CH.sub.3CN
gradient to afford the desired Gd complex (12.67 g; 10.74).
[0300] The purification of the complex on weakly cation-exchange
resin yielded the product with the phenol group deprotonated, as
confirmed by the elemental analysis.
[0301] Yield: 70%
[0302] m.p.: >280.degree. C.
[0303] HPLC assay: 99.5% (in % area)
[0304] Elemental analysis
8 C H Gd I N Na P % calc.: 25.46 2.14 13.33 21.52 3.56 9.75 5.25 %
24.01 3.09 12.44 20.19 3.35 8.52 5.16 H.sub.2O 6.56% found:
[0305] IR and MS spectra are consistent with the indicated
structure.
EXAMPLE 5
[0306] Gadolinium complex of
N,N'-[(phosphonomethylimino)di-2,1-ethanediyl-
]bis[N-(carboxymethyl)glycine] salified with Na (1:3) 22
[0307] A)
N,N'-[[[(Diethoxyphosphinyl)methyl]imino]di-2,1-ethanediyl]bis[N-
-[2-(1,1-dimethylethoxy)-2-oxoethyl]glycine]1,1-dimethylethyl
ester
[0308] The product is prepared by reacting the aminophosphonate
(15.09 g; 90.28 mol) prepared as described in 1F and the bromide
(70.09 g; 199.0 mol) prepared as described in J. Org. Chem. 1993,
58, 1151. The reaction is carried out as reported in 1G and the
product is recovered as a colourless oil (35.59 g; 50.14 mol).
[0309] Yield: 56%
[0310] HPLC assay: 95% (in % area)
[0311] TLC; Carrier: silica gel plate 60F 254 Merck
[0312] Eluent: toluene/EtOAc/iPrOH=50:45:5
[0313] Detection: 0.5% KMnO.sub.4 in 1M NaOH; 254 nm; Rf=0.40
[0314] .sup.1H-NMR, .sup.13C-NM, IR and MS spectra are consistent
with the indicated structure.
[0315] B)
N,N'-((Phosphonomethylimino)di-2,1-ethanediyl]bis[N-(carboxymeth-
yl)-glycine]
[0316] The hexaester prepared at point A (29.23 g; 41.18 mol) is
deprotected with iodomethylsilane, under the same conditions as
reported in 1H. The resulting crude is loaded onto a Relite.RTM. 3
AS/fb column which is eluted with water until elimination of the
residual iodide and then onto a column of Dowex.RTM. CCR3LB resin
maintained at 60.degree. C. The solution comprising the purified
compound is freeze-dried to obtain the chelating agent as a white
solid (9.69 g; 22.6 mol).
[0317] Yield: 55%
[0318] m.p.: 110-114.degree. C.
[0319] HPLC assay: 96% (in % area)
[0320] Elemental analysis
9 C H N P % calc.: 36.37 5.63 9.79 7.21 % found: 35.03 5.76 9.43
6.92 H.sub.2O 1.05%
[0321] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0322] C) Gadolinium complex of
N,N-[(Phosphonomethylimino)di-2,1-ethanedi-
yl]bis[N-(carboxymethyl)glycine]trisodium salt.
[0323] To a solution of the chelating agent obtained as in step B
(6.0 g; 13.97 mol) in H.sub.2O (60 mL) is added 2N NaOH (15 mL) and
Gd.sub.2O.sub.3 (2.53 g; 6.99 mol). The suspension is heated at
70.degree. C. for one hour, then filtered through Millipore.RTM.
HAWP 0.45 .mu.m filter. The solution is neutralized with 2N NaOH
and concentrated to dryness to obtain the title complex as a white
solid (9.1 g; 14 mol) in quantitative yield.
[0324] m.p.: >250.degree. C.
[0325] HPLC assay: 100% (in % area)
[0326] Elemental analysis
10 C H Gd N Na P % calc.: 24.04 2.79 24.21 6.47 10.62 4.77 %
found.: 23.94 3.00 24.06 6.45 10.74 4.71
[0327] IR and MS spectra are consistent with the indicated
structure.
EXAMPLE 6
[0328] Gadolinium complex of
N,N'-[(Phosphonomethylimino)di-2,1-ethanediyl-
]bis(N-(phosphonomethyl)glycine] salified with Na (1:5) 23
[0329] The aminomethylphosphonic acid diethyl ester, prepared as
described in example 1F, is reacted with the bromo derivative
prepared according to Example 4D.
[0330] The resulting hexaester is deprotected under the same
conditions as reported in 4F. The isolated acidic chelating agent
is complexed according to the procedure reported in 3G.
EXAMPLE 7
[0331] Gadolinium complex of
N,N'-[[[3-Carboxy-1-phosphonopropyl]imino]di--
2,1-ethanediyl]bis[N-(carboxymethyl)glycine] salified with
triethylamine (1:4) 24
[0332] A)
2,2'-(Iminodi-2,1-ethanediyl)bis-1H-isoindole-1,3(2H)-dione)
[0333] A mixture of phthalic anhydride (32.0 g; 0.216 mol) and
diethylenetriamine (10.32 g; 0.1 mol) in acetic acid (106 g) is
refluxed for 1 h. The acetic acid is removed on a rotary evaporator
and the pale yellow oil obtained is allowed to stand under vacuum
overnight. The oil solidified on standing and this is then
triturated with a saturated solution of sodium bicarbonate to
remove acetic acid and some unreacted phthalic anhydride. The
yellow solid is then filtered, washed with water, dissolved with
chloroform (500 mL), and the chloroform solution is dried with
Na.sub.2SO.sub.4. Evaporation of the chloroform gave a solid (28.0
g).
[0334] Yield: 77.1%.
[0335] An analytical sample is crystallized from ethanol.
[0336] mp: 180-81.degree. C.
[0337] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0338] HPLC assay: 96.7% (in % area)
[0339] B)
4-[Bis[2-(1,3dihydro-1,3-dioxo-2H-isoindol-2-yl)ethyl]amino]4bis-
(1,1-dimethylethoxy)phosphinyl]butanoic acid phenylmethyl ester
[0340] A solution of the 3-benzyloxycarbonylpropionaldehyde (4.0 g;
0.0208 mol) in acetonitrile (25 mL) is added to a slurry of
bis(phthalimido) derivative prepared at point A) (6.8 g; 0.0187
mol) in acetonitrile (75 mL) over a period of 30 min. The
temperature of the reaction mixture is maintained at 80-90.degree.
C. during the addition. The reaction mixture is stirred at
80.degree. C. for an additional 30 min. The colour of the reaction
mixture turns yellow during the addition of the aldehyde.
Tris(tert-butyl) phosphite (5.2 g; 0.0208 mol) in acetonitrile (15
mL) is added dropwise and the reaction mixture is stirred at room
temperature for 48 h The reaction mixture becomes a clear yellow
solution after the addition of tris(tert-butyl)phosphite (.about.3
h). Acetonitrile is then removed and the residue is treated with
EtOAc (50 mL). The solid formed is filtered and the EtOAc solution
is directly applied to a column of silica gel (packed in 50:50
hexane-EtOAc). The column is initially eluted with hexane-EtOAc
(600 mL) and then eluted with 70:30 (EtOAc:hexane). Fractions
containing the product are collected and evaporated to give an oil.
This is dried under vacuum to give a white solid (9.9 g)
[0341] Yield 72%.
[0342] An analytical sample is crystallised from hexane-EtOAc.
[0343] mp.: 120-121.degree. C.
[0344] HPLC assay: 99.2% (in % area)
[0345] Elemental analysys
11 C H N % calc.: 64.00 6.30 5.70 % found: 63.90 6.34 5.54
[0346] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0347] C)
4-[Bis(2-aminoethyl)amino]4[bis(1,1-dimethylethoxy)phosphinyl]bu-
tanoic acid phenylmethyl ester
[0348] To a solution of tert-butoxyphosphinyl derivative prepared
at point B) (5.65 g; 0.0078 mol) in CH.sub.2Cl.sub.2 (50.0 mL) is
added hydrazine (1.5 g; 0.0468 mol) followed by water (0.2 mL) and
the reaction mixture is stirred at room temperature. The reaction
is followed by .sup.1H NMR (the reaction is complete in 36 h). The
precipitated phthalyl hydrazide is filtered through Celite.RTM. and
the filter cake is washed with CH.sub.2Cl.sub.2. The combined
methylene chloride solution is evaporated to give a thick oil,
which is dried under vacuum. The diamine obtained (3.61 g) is used
for the alkylation step without further purification.
[0349] Yield: 97%.
[0350] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0351] D)
N,N'-[[[1-Bis(1,1-dimethylethoxy)phosphinyl]-3-[(phenylmethoxy)c-
arbonyl]propyl]imino]di-2,1-ethanediyl]bis[N-[(1,1-dimethylethoxy)-2-oxoet-
hyl]glycine 1,1-dimethylethyl ester]
[0352] To a solution of the diamino intermediate prepared at point
C) (7.5 g; 0.0159 mol) in acetonitrile (60 mL) are added
diisopropylethylamine(18- .55 g; 25 mL; 0.142 mol) and tert-butyl
bromoacetate(13.2 g; 10.0 mL; 0.0695 mol) and the mixture is
stirred at room temperature overnight. Acetonitrile and excess
diisopropylethylamine are removed on a rotary evaporator and the
residue is basified with K.sub.2CO.sub.3 solution (5%, 100 mL). The
reaction mixture is extracted with diethylether (2.times.150 mL)
and the diethylether solution is washed with water and dried
(Na.sub.2SO.sub.4). Evaporation of diethylether gives an oil, which
is purified by silica gel chromatography. The column is packed with
hexane-EtOAc (7:3) and eluted with hexane-EtOAc (7:3) (500 mL) and
then with hexane-EtOAc (5:5). Fractions (Rf 0.5) comprising the
desired compound are collected and evaporated to give a thick
viscous oil (7.5 g).
[0353] Yield: 51%.
[0354] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0355] E)
N,N'-[[[1-(Bis(1,1-dimethylethoxy)phosphinyl]-3-carboxypropyl]im-
ino]di-2,1-ethanediyl]bis[N-[2-(1,1dimethylethoxy)-2-oxoethyl]glycine
1,1-dimethylethyl ester]
[0356] To a solution of the benzylester prepared at point D) (4.63
g; 0.005 mol) in THF (30 mL) is added 10% Pd--C (2.0 g, Degussa
type .about.50% water) and the mixture is hydrogenated at 45 psi
for 12 h The catalyst is filtered through Celite.RTM. and the
filter cake is washed with THF (2.times.30 mL). The combined T1F
solution is concentrated on a rotary evaporator to give the acid as
a thick viscous oil. This is dried under vacuum for 24 h isolating
the desired compounds (3.98 g) which is used as such without firer
purification.
[0357] Yield: 95.2%
[0358] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0359] F)
N,N'-[[[3-Carboxy-1-phosphonopropyl]imino]di-2,1-ethanediyl]bis[-
N-(carboxymethyl)glycine]
[0360] The hexaester prepared at step E) (0.84 g, 0.001 mol) is
dissolved in TFA and the mixture is stirred at room temperature for
24 h. TFA is removed on a rotary evaporator and the residue is
treated with anhydrous diethylether (10 mL). The precipitated solid
is filtered and dried under vacuum to give the acid chelating
ligand as TFA salt (0.5 g).
[0361] Yield: 90%
[0362] HPLC assay: 86% (in % area)
[0363] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0364] G) Gadolinium complex of
N,N'-[[[3-Carboxy-1-phosphonopropyl]imino]-
di-2,1-ethanediyl]bis[N-(carboxymethyl)glycine]salified with
triethylamine
[0365] The acid chelating ligand TFA salt obtained at step F) (0.25
g) is dissolved in a mixture of acetonitrile water 1:1 (5 mL) and
Gd (OAc).sub.3 (0.25 g; 0.006 mol) is then added to the reaction
mixture. The initial pH of the reaction mixture is found to be
1.29. The pH of the solution is then adjusted to 5.0 by the
addition of 1N NaOH and the mixture is stirred at room temperature
for 24 h. The Gd complex is then purified by DEAE Sephadex
chromatography using triethylamine bicarbonate buffer. The
fractions eluted at 800-mM buffer are collected and freeze dried to
give the Gd complex as a triethylamine salt (0.2 g).
[0366] HPLC assay: 100% (in % area)
[0367] Elemental Analysis: Calcd. for
C.sub.16H.sub.25N.sub.3O.sub.13PGd.2- .6
C.sub.6H.sub.15N.3.H.sub.2O
12 C H Gd N H.sub.2O % calc.: 39.02 7.25 16.10 8.06 3.00 % found.:
39.09 7.33 16.19 8.25 3.03
[0368] IR and MS spectra are consistent with the indicated
structure.
EXAMPLE 8
[0369] Gadolinium complex of
4-Phenyl-N-[trans-4[[([4bis[2-[bis(carboxymet-
hyl)amino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino]methyl]cyclohexylcarbo-
nyl]-L-phenylalanine, salified with meglumine (1: 4) 25
[0370] A)
4Phenyl-N-[[[trans-4-[[4-[bis[2-bis[2-(1,1-dimethylethoxy)-2-oxo-
ethyl]amino]ethyl]amino]-4
[bis(1,1-dimethylethoxy)phosphinyl]-1-oxobutyl]-
amino]methyl]cyclohexyl]carbonyl]-L-phenylalanine phenylmethyl
ester
[0371] To a solution of the monoacid obtained as disclosed at point
E) of EXAMPLE 7 (0.9 g; 1.075 mol) in CH.sub.2Cl.sub.2 (15 mL) is
added HATU (O-(7-Azabenzotriazol
-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (0.475 g;
125 mol) and the mixture is stirred at 0.degree. C. for 10 min.
Diisopropylethylamine 0.39 g (3.0 mol) is then added and the
mixture is stirred at 0.degree. C. for further 10 min. aB.-TFA salt
[4-Phenyl-N-[[trans-4-(aminomethyl)cyclohexyl]carbonyl]-L-ph-
enylalanine phenylmethyl ester] (0.584 g; 1.0 mol) (the amino
compound 7) is then added to the reaction mixture and stirred at
0.degree. C. for 2 h and at room temperature for 24 h
CH.sub.2Cl.sub.2 is removed and the residue is extracted with EtOAc
(75.0 mL). The EtOAc solution is washed with K.sub.2CO.sub.3
solution (10%, 2.times.50 mL), water, and dried (Na.sub.2SO.sub.4).
Evaporation of EtOAc gives an oil which is dried under vacuum to
give a foamy solid, which is purified by silica gel column
chromatography using EtOAc. Product containing fractions are
collected and evaporated to give an oil, which is dried under
vacuum to give foamy solid (1.1 g). This is found to be
analytically pure and used in the next step.
[0372] Yield: 85%
[0373] Elemental analysis
13 C H N % calc.: 65.10 8.40 5.40 % found: 64.55 7.86 5.31
[0374] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0375] B)
4Phenyl-N-[[trans-4-[[[4-[bis[2-[bis[2-(1,1-dimethylethoxy)-2-ox-
oethyl]amino]ethyl]amino]-4-[bis(1,1-dimethylethoxy)phosphinyl]-1-oxobutyl-
]amino]methyl]cyclohexyl]carbonyl]-L-phenylalanine
[0376] To a solution of the benzylester obtained as disclosed at
point A) (1 g; 0.0078 mol), is added 10% Pd--C (0.5 g; Degussa
type, almost 50% water) and the mixture is hydrogenated at 45 psi
for 8 h. The catalyst is filtered through Celite.RTM. and the
filter cake is washed with THF (2.times.30 mL). The combined THF
solution is concentrated on a rotary evaporator to give the acid as
a thick viscous oil. This is dried under vacuum for 24 h to give a
foamy solid (0.9 g) subsequently used as such without further
purification.
[0377] Yield: 97%
[0378] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0379] C)
4-Phenyl-N-[[trans-4-[[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]-
amino-1-oxo-4-phosphonobutyl]amino]methyl]cyclohexyl]carbonyl]-L-phenylala-
nine
[0380] Trifluoroacetic acid (4 mL) is added to the
hexa-(tert-butyl) ester (0.9 g; 0.75 mol) prepared at point B) and
the mixture is stirred at room temperature for 12 h Diethylether
(30.0 mL) is then added to the reaction mixture and the precipitate
is filtered and dried under vacuum to give a white solid (0.8 g)
(Yield: 97%). HPLC analysis of the TFA salt indicates that it is
fairly pure and useful for Gd chelation without fiercer
purification. An analytical sample is obtained from TFA salt (100
mg) by preparative HPLC. Fractions containing the pure product are
collected and freeze dried to give a white fluffy solid (50
mg).
[0381] Purification yield. 50%
[0382] Elemental analysis (calcd. for
C.sub.41H.sub.55F.sub.3N.sub.5O.sub.- 17P.2H.sub.2O)
14 C H N % calc.: 48.81 6.05 6.84 % found: 48.60 5.90 6.90
[0383] .sup.1H-NMR, .sup.13C-NMR, .sup.31P-NMR, IR and MS spectra
are consistent with the indicated structure.
[0384] D) Gadolinium complex of
4-Phenyl-N-[trans-4-[[[4-[bis[2-[bis(carbo-
xymethyl)amino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino]methyl]cyclohexyl-
carbonyl]-L-phenylalanine
[0385] TFA salt obtained as disclosed at point C (0.3 g; 0.275 mol)
is added to a mixture of acetonitrile (7.0 mL) and water (2 mL). To
this a solution of Gd(OAc).sub.3 (0.132 g; 0.325 mol) in water.
(2.0 mL) is added dropwise. The initial pH of the solution is found
to be 1.29. The solution becomes turbid and the pH of the solution
is adjusted to 5.0 by adding a solution of meglumine in water. The
turbid reaction mixture is stirred at room temperature for 48 h The
pH of the reaction mixture is then raised to 9.0 by the addition of
meglumine solution and then purified by preparative HPLC using
acetonitrile and water. Fractions containing the pure product were
collected and freeze died to give a fluffy solid (220 mg).,
[0386] Yield: 78.5%
[0387] HPLC assay 97.7% (in % area)
[0388] Elemental analysis for
C.sub.67H.sub.19N.sub.9O.sub.35PGd.5H.sub.2O
15 C H N Gd % calc.: 42.60 6.88 6.67 8.32 % found: 42.54 6.93 6.47
7.06
[0389] IR and MS spectra are consistent with the indicated
structure.
EXAMPLE 9
[0390] Gadolinium complex of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[4-[b-
is[2-[bis(carboxymethyl)amino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino]-7-
,12-dihydroxycholan-24oic acid, salified with sodium (1:4) 26
[0391] A)
(3.alpha.,5.beta.,7.alpha.,12.alpha.)-3-[[4-[bis[2-[bis[2-(1,1-d-
imethylethoxy)-2-oxoethyl]amino]ethyl]amino]-4-[bis(1,1-dimethylethoxy)pho-
sphinyl]-1-oxobutyl]amino]-7,12-dihydroxycholan-24-oic acid methyl
ester
[0392] To a solution of the monoacid obtained as disclosed at point
E) of EXAMPLE 7 (1.2 g; 1.4 mol) in CH.sub.2Cl.sub.2 is added HATU
(0.6 g, 1.5 mol) and the mixture is stirred at 0..degree. C. for 10
min. Diisopropylethylamine (1 mL) is then added and the mixture is
stirred at 0.degree. C. for further 10 min. 3.beta.-Aminocholic
acid methyl ester (compound disclosed in WO 9532741) (0.526 g, 1.25
mol) is then added to the mixture and stirred at 0.degree. C. for 2
h and at room temperature for 48 h. CH.sub.2Cl.sub.2 is removed on
a rotary evaporator and the residue is extracted with EtOAc. The
EtOAc layer is washed with K.sub.2CO.sub.3 (10%, 2.times.30 mL),
water and dried (Na.sub.2SO.sub.4). Evaporation of the EtOAc gives
an oil which is purified by silica gel column chromatography
(CH.sub.2Cl.sub.2:CH.sub.3OH, 95:5). Product containing fractions
(Rf=0.5,) are collected and evaporated to give an oil which is
dried under vacuum to give the desire compound (1.1 g) as a foamy
solid.
[0393] Yield 71%.
[0394] TLC: Carrier: silica gel plate 60F 254 Merck
[0395] Eluent: CH.sub.2Cl.sub.2:CH.sub.3OH 95:5
[0396] Detection: I.sub.2, 254 nm 0.5
[0397] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0398] B)
(3.alpha.,5.beta.,7.alpha.,12.alpha.)-3-[[4-[bis[2-[bis[2-(1,1-d-
imethylethoxy)-2-oxoethyl]amino]ethyl]amino]-4-[bis(1,1-dimethylethoxy)pho-
sphinyl]-1-oxobutyl]amino]-7,12-bis(acetyloxy)cholan-24oic acid
methyl ester
[0399] To a mixture of pyridine and acetic anhydride (1:1, 2 mL) is
added the dihydroxy cholic acid derivative prepared at step A)
(1.24 g; 1 mol) and the mixture is stirred at room temperature for
48 h. Excess acetic anhydride and pyridine are removed and the
residue is treated with a saturated solution of sodium bicarbonate
(5.0 mL) and extracted with diethylether. The diethylether solution
is washed with water and dried (Na.sub.2SO.sub.4). Evaporation of
diethylether give a viscous oil which is purified by silica gel
column chromatography using EtOAc-hexane (8:2). Fractions
containing the product are collected and evaporated to give an oil
which is dried under vacuum to give the desired diacetate (0.44 g)
as a foamy solid.
[0400] Yield 75%.
[0401] .sup.1H-NMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0402] C)
(3.alpha.,5.beta.,7.alpha.,12.alpha.)-3-[[4-bis[2-[bis(carboxyme-
thyl)amino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino]-7,12-dihydroxycholan-
-24oic acid
[0403] Trifluoroacetic acid (3 mL) is added to the hexa
(tert-butyl) ester prepared at step 1) (0.662 g; 0.5 mol) and the
mixture is stirred at room temperature for 30 min and kept at
4.degree. C. for 18 h. Trifluoroacetic acid is then removed to give
the desired deprotected intermediate (0.52 g) as an oil which is
dried under vacuum. To a solution of said compound in ethanol water
(1:1,5 mL) is added sodium hydroxide (20%, 5 mL) and the mixture is
stirred at room temperature for 24 h. The completion of the
reaction is followed by HPLC. The solvents are removed and the
residue is neutralized with 1N HCl to give the completely
deprotected polyacid as hydrochloride, which is filtered and dried
under vacuum to give the desired chelating compound (0.35 g).
[0404] Yield 79%.
[0405] HPLC assay: 98% (in % area)
[0406] .sup.1HNMR, .sup.13C-NMR, IR and MS spectra are consistent
with the indicated structure.
[0407] D) Gadolinium complex of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[4-
-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino-
]-7,12-dihydroxycholan-24-oic acid, salified with sodium (1:4)
[0408] To a solution of the GdCl.sub.3.6 H.sub.2O (50 mg, 0.135
mol) in water (2 mL) is added the hydrochloride prepared at step C)
(0.104 g; 0.1 mol) dissolved in a mixture of acetonitrile-water
(1:1; 7 mL). The initial pH of the reaction mixture is found to be
1.27. The pH of the reaction mixture is raised to 5.5 by the
addition of 1N NaOH. The turbid reaction mixture is stirred for 48
h and the pH of the solution is raised 10 by the addition of 1N
NaOH. The turbid solution obtained is centrifuged, the clear
supernatant solution is collected and purified by preparative HPLC.
Fractions containing the pure product are collected and freeze
dried to give the desired compound.
[0409] Yield: 62%.
[0410] HPLC assay: 98% (in % area)
[0411] Elemental Analysis: (for
C.sub.40H.sub.60N.sub.4O.sub.16PGd.4Na.8.H- .sub.2O)
16 C H Gd N % calc.: 37.62 6.00 12.31 4.39 % found.: 37.37 5.95
11.43 4.40
[0412] MS: (M+H).sup.+=1046.4
[0413] IR spectrum is consistent with the indicated structure.
* * * * *