CA2115275A1 - Amino acid, ester and/or catechol contrast agents for mri - Google Patents

Abstract

The present invention relates to the preparation of amino acid containing compounds which also have multiple carboxylic acid functional groups. Paramagnetic metal (II) or (III) ion chelate complexes are formed using these compounds for use as intravenous contrast agents to produce enhanced contrast magnetic resonance images of the heart, liver, biliary tree or upper small intestine. The mono and di-amino acids (and their carboxylic esters carboxylic amides, and catechols) of EDTA, DTPA, and the like are prepared.
The paramagnetic metal (II) or (III) ion complexes are formed and produce T1-related contrast effects in MR images. The compounds and complexes also appear to have low toxicities and to be relatively rapidly and completely cleared from the tissue of a living mammal, e.g. a human being.

Description

16 ~c~P~T~P I O 0 9 ,~A~ 1993 !~ , . 92 / 0 6660 AMINO ACID, ESTER ANDIOR CATECHOL ~113 ~ 7 5 CON~RAST AGENTS FOR MRI
BACKGROUND OF ~HE INVENTION
Related AppLic~tions This application is a Continuation-In-Part of U.S.
Serial ~o. 744,470~ filed August 12, 1991 which is a Continuation-In-Part of U.S. Serial No. 743,143, filed August 9, 1991.
~ield of the Invention The present inventi~n re}ates to the preparation and use of amino acid and catechol containing hepatobiliary and cardiac contrast agents useful in magnetic resonanc~
imaging. The contrast agents have multiple carboxyl groups to chelate a variety of metal (II) or (III) ions. -~escription of Related Art This invention relates to contrast agents for medicalmagnetic resonance imaging (MRI).
A contrast agent is an exogenous substance that either auqments or suppresses the normal in vivo MRI si~nal, thereby yielding additional diagnostic information. The theory (1,2) and applications of various types o~ contrast agents has been described in the literature (1,2). The Arabic numbers in parentheses in this section refer to the articles cited in this section.
; ~ ~ 25 The applications of a given MRI contrast agent are determined by its distribution in vivo. The mechanisms controllinq the initial biodistribution can be classed as physi~o-chemical, i.e., dependent on}y upon such properties as molecular size, charge, lipophil~city, surface -properties, etc., or receptor-mediated--dependent upon the binding o~ a substrate to a specific receptor in or on cells. Different orqan~ may handle the same contrast agent by different mechanicms. For example, the mole~ular size of the agent may result in its filtration by the kidneys ~or con~inement to the ~ascular space) while it is cleared au~ S HEET

-` 16 R~td PCT/PTO O 9 MAR 199;
PCT~US 92~0666( by receptor-mediated transport in the liver. 2 11 52 7a Contrast agents exhibiting a physico-chemical distribution mechanism include th2 gadolinium (III) complex of diethylenetriaminepentaacetic acid (Gd-DTPA), which distributes in plasma and extracellular fluid, and albumin-(Gd-DTPA~, which remains largely intravascular (1,2)~. The former is used to demonstrate blood-brain barrier lesions or to reveal renal anatomy and function (3), while the latter has been used experimentally to delineate the ~asculature (4) and determine brain blood volume (5,6).
Iron-dextran, although a colloid, has a sufficiently long plasma half-life (12 hr) to be used.as an intravascular T2 contrast agent (7), as do some superparamagnetic iron oxide particle preparations (8,9). ~`
lSBecause of its role in the removal of exogenous compounds from general circulation, the liver is able to acti~ely take up and concentrate soluble, as well a~
;~ particulate, contrast agents. The pathways followed by solutes from plasma to bile have been reYiewed (10-11) and are diagrammed in Figure 4. Passage into the hepatocyte across the cell membrane can take place by pinocytosis, passive diffusion, and/or by carrier-mediated systems that transport bile aci~s, bilirubin, organic anions, organic cations, neutral organic compounds, or inorganic ione. The substrate specificity of different carrier systems can partially overlap (e.g., organic anions and bile acids).
The substrate may be metabolized intracellularly and/or conjugated with glucuronic acid or glutathione, for example. Finally, excretion into bile canaliculi again 30 ` involves passage through a cell membrane. The mechanism of biliary excretion for a gi~en compound ma~ differ from that _ operative for its uptake.
The relative rates of metabolism, biliary elimination, and renal excretion determine the clearance of drugs and théir metabolites from plasma and their pers~stence in any SUBSTlTUrE SHEET
IPEA US

16 Rec'd PGT/PT0 0 9 MAR ~993 ` ~C~/vs 9~/06660 3 2~ 1~27~
one organ system. However, presently the factors that direct one compound to be excreted in the bile and another in the urine are not completely understood. Molecular weight, polarity, and molecular structure in relation to binding to plasma and transporter proteins are important.
- There appears to be a general molecular weight threshold, which is species-dependent (ca. 300 for rats and 600 or humans3 below which urinary excretion dominates (10-11).
Hydrophilic-lipophilic balance appears to play a critical role in biliary excretion (26-28). However, a priori prediction is not presently possible.
The liver has provided the first example of receptor-mediated localization of an MR contrast agent -- Fe-EHPG
(EHPG is Ethylene-bis(hydroxyphenylglycine)) (12).- Other iron (13-153, manganese (16-17), and gadolinium (18) chelates have since been described that have either potential for, or have demonstrated receptor-mediated hepatocyte uptake. -~ It has been reported by others that the anionic ;~ ~ 20 chelates Fe-EHPG, Fe-HBED tHBED = bis-(hydroxybenzyl) ethylenediaminediacetic acid), and Fe-PGDF ~PGDF = N-3-(phenylglutaryl)desferrioxamine B) are transported in the - liver by a system or systems inhibitable by BSP
(bromosulfophthalein) (13 ,15) .
, 25 The lipophilic chslate Gd-BOPTA ("benzyloxypropionic-tetraacetate," a derivative of DTPA) was shown to have significant biliary excretion (38.6% of injected dose in blle at 6 hr) (18). No information was reported on the mec~anism of transport ~e.g., passive diffusion or anionic i~ transport) of this compound. Gd-BOPTA produced a larger signal enhancement (48%) in liver than Gd-DTPA (16%) in Tl-;~ weighted spin-echo imag~s at 0.5 Tesla.
Additionally, other organs and tissues may posses receptors with affinity for certain classes of 6ubstrates, e.g., amino acids, peptides or catechol amines (19-24).

~UI~TITI~E SHEEr '' IPEA~US

16 R~'d PCT/PT0 0 9 MAR 1~9 3 T/US 9~06660 4 21~27~
These receptors may also bind molecules that resemble the substrate, e.g., a derivative of an amino acid that is - present in a peptide substrate (22) or an amide derivative of a naturally occurring catechol amine such as dopamine.
The contrast agents of this invention may in part localize by such a mechanism. Furthermore, the localization of the catechol containing contrast agents of the pr~sent invention may depend in part on their respective reduction-oxidation properties.
10 To date, magnetic resonance imaging (~RI) has played a minor role in imaqing of the liver and a~domen of a human being because of degradation of image quality by motion artifacts, and by the lack of suitable contrast agents.
Recent technical advances in instrumentation (e.g., self-shielded gradient coils~ and pulse ~equences (e.g., echo-planar and turbo-flash techniques~ promise to alleviate the motion-related problems of the torso and abdomen, and make contrast agent development all the more important for continued progress in abdominal MRI.
General background in the use of MRI contrast agents and of their preparation and purification are described, for example, in:
H. Gries et al., U.S. Patent No. 4,647,447;
: ~ R.B. Lauffer et al., U.S. Patents 4,899,755 and 2~ 4,880,008;
B.L. Engelstad et al., U.S. Patent 4,909,257;
.L. White et al., U.S. Patent 4,999,445.
1. R.B. Lauffer, "Paramagnetic metal complexes as water pro~on relaxation agents for NMR imaging: Theory and de~ign," Chem Rev. (1987); 87:901-927.
2. S. M. Rocklage, et al.- "Contrast Agents in Amgnetic re50nance imaging." Chapter 14, In Maqneti~
Resonance Imaqina, 2nd ed, Stark DD, Brad~ey WG, eds. St.
Louis: C~V. Mosby Co. (1992).

3. G. Bydder, "Clinical applications of Gadolinium-SUBSTrrUrE SHEET
i' ~EA~S

- 16 ReG'd PCT/PTO O 9 M~R ~993 ~CT~ ~j S 92 ~ 0 6660 211~27~
DTPA." In Maqnetic Resonance Imaqing. Stark DD, Bradley WG, eds~ St. Louis: C.V. Mosby Co. tl988); 182-200 (Chap.
10~ .

4. M.E. Moseley et al., "Vascular mapping using - 5 Albumin-(Gd-DTPA), an intravascular MR contrast agent, and projection MR imaging," J. Computer Assist Tomoaraphy ~1988); 13:219-221.
5. T.A. Kent et al., "Cerebral blood volume in a rat model of cerebral ischemia by MR imagin~ at 4.7 T," ~J~R
10 (1989~; 10:335-358.

6. D.L. Whit et al., "Determination of perfused cerPbral blood volume using an intravascular MR contrast agent," Book of Abstracts: Society of Magnetic Resonance in Medicine (1989); 2:806.

7. D.L. White et al., "Iron-Dextran as a magnetic susceptibility contrast agent: Flow-related contrast effects in the T2-weighted spin-echo MRI of normal rat and cat brain," Maqn.Reson.Med. ~1992), Vol. 24 , p.14~

8. D.L. White et al., "Plasma clearance of ferrosomes, a long-lived superparamagnetic MRI contrast : agent. Book of Ab~stracts: 5Ociety of Magnetic Resonance :~
in Medicine (1990); 1:51.
. R. Weissleder et al., "Ultrasmall superparamagnetic iron oxide: Characterization of a new 2~ class of contrast aqent for MR imaging, "Radiology (1990);
~:~ 175:489-493.
10. L.S. Schanker, "Secretion of organic compounds into bile. In The Handbook of Ph the st~ucture physlology.
~limentary Canal V. Washington, D.C.: American Physiol.
30 Society, Chap. 114:2433-2449.
~:~ 11. C.D. Klaassen et al ., "Mechanisms of bile formation, hepatic uptake, and biliary excretion," Pharm~
~y (~984); 36:1-67.
12. R.B. Lau~er et al., "Iron-E~PG as a hepatobiliary MR contrast agent: Initial imaging and UBSTITUTE SHEET
IPEAIUS

16 Rec'd PCT/PT0 0 9 MAR i9 ~11S 9~Q1~660 2, 1~27~

biodistribution studies, J. Computer Assist Tomograp~, ~1985); 9:431-438.
13. B. Hoener et al., "Evaluation of Fe HBED and Fe-EHPG as magnetic resonanoe contrast agents for assessing hepatobiliary function, J. Magn. Reson Imaqinq, tl9gl);
1:357-362.
14. K.A. Muetterties et al., 9'Ferrioxamine B
derivatives as hepato~iliary contras~ agents for magne~ic resonance imaging, Ma~n Reson Med (1991); Vol. 22, pp. ~8 ~o 100.
15~ B. Hoener et al., "Hepatic transport of th~
magnetic resonance imaging contrast agent F~(III)-N-(3-Phenyl-glutaryl)desferrioxamine B. Ma~n R son Med. (1990);
17:5~9-51.
1~. ~.L. White et al., "Clearance, excretion, and organ distribution of a new MRI contrast agent Manganese-Dipyridoxal-Diphosphate (Mn-DPDP). Abstract Book: Society of Magnetic Resonance in Medicine (1988~ 1:531.
17. S.W. Young, "MRI measurement of hepatocyte ZO toxicity using the new MRI contrast agent manganese dipyridoxal diphosphate, a manganese/pyrdoxal 5-phosphate chelate," Maq Reson Med. (1989); 10:1-13.
18. P. Pavone et al., "Comparison of Gd-BOPTA with Gd-DTPA in MRI imaging of rat liver," Radiolo~y ~1990~;
17Ç:61-640 19. P. Ascher, "Glutamate receptors and glutamatergic synapSes. In Receptors, Membrane TranspQrt a~d Sianal Transduction. A.E. Evangelopoulis et al., Berlin:!Springer Verlag. (1989): 127-146.
20. F.P. Lehman, "Stereoselective Molecular Recogni~ion in Biology. In Receptors and Recoani~ion, Vol.
~, Series A. Cuatrecasas P and Greaves MF.
London:Chapman-Hall (1978).
21. R.D. O'Brien, ed. ThB Receptors. A Çom~re~*nsive reati5e, Yol. 1, New York:Plenum Press (197~.

IpEAnJs - 16 Rec'd PCTIPTO O 9 MA.~ ~9 I~CT/ U S 92 / 0 6660 211~27~
22. S.S. Schiffman et al., "The Search for Receptor that Mediate Sweetness," In The Receptors, Vol. 4, Conn PM, ed. Orlando:Academic Press (1986).
23. A. S. Horn, et al., eds. The Neurobiolo~y of Dopamine. A~ademic Press. New York. 197g.
24t B. J. Clark. "The role of dopamine in the periphery," in The ~opaminergic Sy~tem, B. Halasz, et al., : eds., Springer-Verlag, Berlin, 1985, p 27-39.
All references articles, patents, etc. citsd in this application are incorporated herein by reference in their : entirety.-It would be very useful to have organic chelate metal ion complexes which are specific for MRI imaging of the liver, the biliary tree, the upper small intestine,~or the ~ 15 myocardial tissue. The present in~ention provides :; complexes and methods having these use~ul ~dvantage~.

The present invention relates to a magnetic resonance ~ imaging contrast agent, comprising the complex:
:~ 20 ~ L-M
wherein M i5 a metal (II) or (III) ion independently selected from the group consisting of metals -: of atomic number 21 to 31, metals of atomic number 39 to 50, the lanthanide metals having an atomic number from 57 to 71, and metals of atomic number 72 to 82, and :~ L is a polydentate organic chelating moiety of structure Ia:

,. . .
: 3~ ~ Q /Y
N- (CH2CH2 ~N)m ~ (CH2CH2N~n C~2CH2N j ~-~ ~
J . X X' Z
wherein ~ 35 Q, J, X, X', Y and Z are each indspendently selected SUBSTITUTE S~IE~T
IPEA/US

- 16Re~'dPCTlPT0 ~9MAR199~
i ~T/ US ~2 ~ 0 66~0 8 21~7~
from DCH2 (C=O) -OH, or -CH2(C=O)NHCH-(R)-(A);
~ herein R in each of Q, J, X, X', Y and Z is independently selected from hydrogen or an or~anic structure comprising an alkyl, aromatic, substituted aromatic, alkylene aromatic, alkylene substituted aromati~, heteroaromatic, substituted heteroaromatic, alkylene heteroaromatic, or alkylene substituted heteroaromatic group provided that at least one of Q, J, Y and Z is -CH2(C=O)-NHCH(R)-A; and ~0 A is independently selected from - (C80) OR~, - rc~o) -N- :
(R2)R3, or R~ wherein R5 is independently selected ~rom -CH2-aryl, -CH2-substituted aryl, -CH2CH2-aryl, or -CH2CH2-substituted aryl, provided that when A is -(C=O)ORI~and R
is hydrogen, then R is not hydrogen;
Rl, R2 and R3 when present in A in each of Q, J, X, X', Y and Z are independently selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl; and m is selected from 0, 1, 2 or 3, and n is selected from 0 or 1, or the pharmaceutlcally acceptable salt(s) thereof.
In another aspect, the present invention relates to a polydentate organic chelating compound of structure I:
Q ," y ~ N-~CH2C~2 ~N)m -(CH2C~2N~n CH2CH2N \
X X~ Z
wherein .
Q, J, X, X', Y and Z are each indep~ndently selected from -CH2(C=O)-OH, or -CH2(C=O)NHCH-(R)-(A);
~ R in each of Q, J, X, X', Y and Z is independently selected from hydrogen or an orga~ic.structure comprising an alkyl, aromatic, substituted aromatic, alkylene aromatic, alkylene substituted aromatic, heteroaromatic, substituted heteroaromatic, alkylene hetQroaromatic, or alkylene substituted heteroaromatic group provided that at least one of Q, J, Y and Z is -CH2(C=O)-NHCH(R)-A; and r;
c .r~
i7 ~.

16 ~e~'d PCT/PTO O 9 MAR 1993 ~ T/US 92 /06660 9 2 1 ~ 7 3 A is independently selected from -(C=O)ORI, -(C=o) -N-(R2)R3, or R4 wherein R4 is independently selected from -CH2-- aryl, -CH2-substituted aryl, -CH2CH2-aryl, or -CH2CH2-substituted aryl provided that when A is -(C=O) ORI and R
is hydrogen then R is not hydrogen;
Rl, R2 and R3 when present in A in each of Q, J, X, X', Y and æ are independen~ly selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl; and m is selected from 0, 1, 2 or 3, and 10 n is selected from O or 1, or the pharmaceutically acceptable salt(s) thereof~
In another aspect, the present invention also relates to a method of preparing a chelate compound of structure (Ia), which method comprises:
(a) contacting a structure of the formula II:
o=C -CH2 CH2 - C-O
o N-~CH2C~2 ~N)m _(CH2CH2N)nCH2CH~N\
-~ O=C -CH2 A D CH2 C=O
: wherein ~ 20 A and D are each -CH2(C=O)-OH, :~ with an amino acid, ester or NHtCH2CH2 aryl or substituted aryl as defined herein:
' H2N-CH (R) -S:~OORI
, wherein R is an organic structure comprising an alkyl, :~ 25 aromatic or a heteroaromatic group, and Rl ls selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl, and m is-selected ~rom 0, 1, 2 or 3, and ~ n is selected from O or 1, in an anhydrous polar aprotic solvent at between about 50 and 150~ ~or between about 2 and 10 hr; and ~b) removing the solvent and recovering the compound of ~tructure I.
These metal ion chelates produce Tl contrast effects in the heart, liver, biliary tree, and upper small SUBST~UTE SHET

16 Rec'd P~T/PTO O 9 MAR 1993 P~T/ US 92 / 0 6660 2il~ ?7a intestine. They demonstrate function, as well as anatomy.
These contra--t agents have low toxicities, and unlike iron - from superparamagnetic particulates, the metal from these compounds should be rapidly and relatively completely cleared fro~ the body. Therefore, these contrast agents are of substantial signific nce to useful zbdominal MRI.
BRIEF DESCRIPTION OF THE ~URES
Figures lA, lB, lC and 1~ ars each a representation of the structur~s of the compounds BOPTA, BSP, DPDP and DTPA, respectively.
Figures 2A, 2B, 2C and 2D are each a representation of the structures o~ the chelates EDTA, EDTP, EHPG and HBED, respectively.
Figure 3 is a representation of a species of the general reaction to produce a bis amino acid substituted chelate.
Figure 4 is a cross-sectional representation of the cell~, componsnts and pathways found in the h~patobiliary region.
Figure 5A is a photograph of T-l weight~d magnetic resonance image5 of a rat at various times (indicated in . minutes) aft~r injection of the Gd-DTPA-(bisphenylal~nine).
: Approximately 0.1 mmol/kg do~e.
Figure 6A is a photograph of T-1 weighted magnetic re.onance images at various times (indicated in min) ob~ained as in Fi~ure 5A and Figure SB for the Gd-DTPA-bis ~phen~-lalanine ethyl ester).
Figs. 5B and 6B are photographic enlargements of the .\ pre- ana 0-min post injection images of Fig. 5A & 6A, resp.
Figure 7 is a photograph of the T-1 weighted magnetic resonance images of two mice side-b~-side at various ti~es (indicated in ~in) after simultaneou~ injection of Gd-DTPA-bisphenyl-alan$ne (bis acid) described in Example 3 below, at approximately 0.1 mmol/kg dose. The images are ~ ~m thick slices in a coronal plane at the levsl of the heart~
;~ BSIIrUTESH~

16 Rec'd PCT/PTO O 9 MAR 1993 P~T/US 92~0666( 211~7J
The heart, liver and intestines are evident.
Figure 8 is a photograph of T-1 weighted magnetic resonance images as obtained for Figure 6 except that a different preparation of Gd-DTPA bis-(phenylalanine ethyl 5 ester) was employed. --Figures 9-13 are each a graphic representation of MRI
imagin~ iA heart, lung, kidney, liver and skeletal muscle tissue, respectively, showing ~ enhancement versus time tmin) for Gd(III)-DTPA-(3HTA)2 and for Gd(III)-DTPA-(DMPE~2, Figure 14A is a photograph of ~-1 weighted MRI images of a rat as obtained (as indicated in min) for Figure 5 using Gd(IIT)-DTPA-(3HTA3~.
Figure 14B is a photograph of a second coronal plane ~.
at the level of the kidneys, as shown in Figure 14A.
~: 15 Figure 15A is a photograph of T-l weighted MRI images of a rat as obtained (as indicated in min) for Figure 5 using Gd(III)-DTPA-(DMPE)2.
Figure 15B is a photograph of a second coronal plane ~; at th~ level of the kidneys, as shown in Figure 15A.
`2Q ~ Figures 16-20 are each a graphic representation of MRI
: imaging in heart, l~ng, kidney, liver and skeletal muscle tissue, respectively, showing ~ enhancement versus time (min) for Gd(III)-DTPA-(L-PheOEt)2and for Gd(III)-DTPA-(D-PheoEt) 2 ' 25 Figures 21A and 21B are each T-1 weighted MRI
photographic images of a rat as obtained for Figures 14 and 15 us~ng Gd(III)-DTPA-(L-PheOEt)2 and for Gd(III)-DTPA-(D-PheO~t)2, ~owever, the dose level was Q.05 mmol/kg.-: .~ on Figures 9 to 13 and 16 to 20 solid vertical lines : 30 within the graph are shown ending in a horizontal line.
The center box of this vertical line is the average for the observation at that point. The hori~ontal lines at eith~r end of the vertical line are located at one standard deri~ation ~rom the center value.

IPEA US

r~ ""~" ",,~,",_, , ,",, ,",, ~ ,, ,",;~ ,,,,,,, ,,", = ,;", " ~ ,"~,,,,,~ ", ,;~ ,~", `` 16 Rec'd PCT~PT0 0 9 MAR 1993 ~C~/ U~ 9~ /06660 12 211~27~
DETAXLED DESCRIPTION OF THE_INVENTION
AND PREFERRED EMBODIMENTS
Definitions As used herein:
"Alkylene" refers to methylene, ethylene, propylene, and the like up to six carbon units.
"Amino acid" refPrs generally to the type of ~-amino acids found in living subjects or mammæls. However, synthetic a-amino acids which ar~ not found in nature are 10 also use~ul. Further these D- and 1- amino acids a-~
separ~te chiral isomers are independently useful. ~ixtures of the D- and L- isomers are also contemplated in this invention.
"Metals of atomic number 21 to 29" refers to candium, 15 titanium, ~anadium, chromium, manganese, iron, coba}t, nickel, copper, zinc and gallium respectively.
Paramagnetic ions are especially preferred. Iron, manganese, nickel, chromium, cobalt are preferred.
"Metal (lanthanides) having an ~tomic number from 57 20 to 71~9 refers to lanthanide, cerium, praseodymium etc. to lutentium, respectively. Paramagnetic gadolinium (III) or dysprosium (III) are preferred.
The contrast agents of this invention localize in several organ systems, e.g., in the kidney, urinary tract, and urinary bladder; in the liver, biliary tree, and intestinal lumen; and in the myocardium. This localization results in increased MRI signal and image contrast. The resulting images show both improv~d anatomic de~aiI and - allow the fùnctional state of certain organ systems, e.g., 30 the urinary and biliary system3, to be ascertained.
This localization ~robably involYes a combination of~
physico-chemical and receptor-based mechanisms. For example, binding to blood components results in enhancement of the blood pool and ~ay contribute to heart enhancement.
Localization in the liver may result from recognition and ; iP~" ~ s~ H~^-ET
.'3~

-` 16 Rec'd PCTIPTO ~ 9 MAR 1993 ~CT~ U S 92 / 0 6660 13 211527~
transport by hepatocytes. Other mechanisms may also be involvPd~ It may be possible to target other organs and tissues by selective modification of the structure of the metal chelate contrast agent.
PreParation of t e amino acid-containinq chelate (L) !having An Ester Group) The following is a general ~escription of the synthesis of the chelating ligand L. Specific descriptions are found in the Experimental Section. :
10In the synthesis of the compounds of structure I, the precursor can be DTPA-bis anhydride (or a si~ilar structure, e.g. EDTA-bis anhydride) which cont~cted with an amino acid of the structure of the known natural or synthetic amino acids, e.g. D, L, or mixtures thereof.
Generally, only one amino acid residue is added to one or more of the locations designatad by Q, J, X, X~, Y or Z, i.e., polypeptide bonds are usually not formed.
With the bi~-anhydride, if a limited amount ~e.g. 0.5 :~ eguivalent) of the amino acid is used, production of the mono amino acid derivative is favored. If two equivalents of amino acid is used, then the bis-amino acid derivative is produced. For ~TPA or higher analogs of polycarboxylic acids, forcing conditions, such as using a coupling reagent ~ and a large excess of the amino acid or protectad amino : ~ 25 acid may be required.
Any anhydrous dipolar aprotic solvent can be used for the synthesis. Dimethylformamide (DMF), dimathylacetamide, acetonitrile or the like are useful. DMF i5 preferred.
The reaction mixture is heated at 70 to 100C for between 3 ~ about 2-12 hr, preferably between 90 and 100C for 4-5 hr, especially 6 hr.
The reaction mixture is cooled and the solvent is removed using a conventional ro~ary ~vaporator or its equivalent. In one aspect, the present invention relates to a novel preparation of the compounds of structura I.

SUB~TITU~E SHEE~
IPEA US

~;~ it~C~ P~ . O ~ 993 ~c'r~ 92 ~0 666 14 2 1 1 ~ 2 7 ~
Preparati~n of the metal ion c~elate complex (L-M~
The general description of the preparation of the chelate metal ion complex, L-M is conventional art. Refer to the references above.
Metal chelates are typically prepared by the reaction of a metal salt or oxide with the chelating ligand in a suitable aqueous or orqanic solvent in the appropriate stoichiometric ratio. Elevated temperatures are sometimes required. The pH of the reaction mixture is then adjusted with a base to obtain the correspsnding chelate ~alt, Alt~rnatively, acid can often be used to obtain the protonated chelate.
The R group preferred as independently selected from an aromatic group, an alkylene aromatic group, a }S substituted aromatic group or a hsteroaromatic group.
Especially preferred are the aryl aromatic groups shown below:

C1l2 ~ OH

: CH2 ~2 ' t--N
H H
.

SUBSmUTE SHEEr IPEA/IJS

16 R~c'd PCTlPTO ~ 9 MAR l9~:
~T/US 92 /06660 ~ 7 ~

The Rl group of the chelating ligand L in each of Q, J, X, X', Y and Z is independently selected from H (the acid), alkyl having from 1 to 7 carbon atoms (the mono, di, tri, etc. acid ester) cyclic groups such as cyclohexyl, phenyl, benzyl or 1- or 2-naphthyl.
Paramagneti~ metal ions are preferred, especially iron (II3 and (III) and gadolinium ~III).
Amino Acid-Amide Structures In another embodiment the present invention relates to spe~ific structures wherei~ A i5 independently ~elect~d from -(C=o)N(R~)R3 wherein R~ and ~ are each independently selected from the group defined for Rl.
General_~ynthesis The amides and related structures ~free amide, mono substituted amide sr disubstituted amide) are produced by tarting with the appropriate amino acid amide (usually as the hydrochloride).
Some purification of the amino acid may be needed.
The amino acid amide is then contacted with the corresponding dianhydride as is described above for the amino acid ester. If a less than equivalent amount of amino acid amide is used and at high dilution in the solvent the mono amino acid amide is favored. If a stoichiometric excess of the amino acid amide is used the diamino acid amide structure is obtained.
Amide structures are also described in Examples 12 to 22. The amide structure~ are u~eful in MRI, because they have gQod contrast properties for specific tissuè and havs ..
~ a longer useful half-life in a mammalian system.
SUBSTTTUTED ALKYLENE ARYL DERIV~T~V~S
In another aspect the present invention relates to sub-~tituted alkylenearyl derivatives, ~e.g~ methylene cat~chols) of EDTA and DTPA-type structures.
The aryl and substituted aryl groups are defined as part o~ group ~. When the NH2CH2CH2-su~stituted aryl is S~ STITUTE SHEE~
IPF~

16 R~'d PCTlPTO O 9 MAR 1993 ~
~CT/ U S 92 ~ 0 666~ :
16 211~27~
contacted with tha bisanhydride as described for the corresponding amino acid ester or amide, the expected compound is obtained. When the substituents on the aryl group are hydroxyl, aqueous base should be avoided.
More specifically the present invention also concerns the preparation of a chelating ligand that bears one or more catecholamide groups, making a stable chelate of tbis ligand with a useful metal ion, and using the chelate for diagnostic imaging or spectroscopy. If the metal ion is paramagnetic, e.g., Gd(III) or Dy(IIX), t~e chelate can produce contrast enhancement in an MRI, or cause shifts, broadening, or other changes in a magnetic resonance spectrum. - ~
These novel agents constitute an improvement over the prior art in that they tend to be lscalized in certain types of tissue by virtue of their resemblance to naturally occurring catecholamines and/or their redox and other physicochemical properties. In particular, two deri~atives of dopamine (also 3-hydroxytyramine or "3-HTA"), DTPA-bis(3-hydroxytyramide), and DTPA-bis(3,4-~ dimethoxyphenethylamide), are useful. These ligands were -~ rea ted with GD(III) to produce the chelates, DTPA-(3-HTA)2 and GD-DTPA-(3,4-DMPE) 2t respectively. These were used as contrast agents in the MRI of rats as described in the Examples. Both chelates demonstrated useful enhancement of heart, lungs, kidney and liver. However, the former s~lectively enhanced the heart.
Maanetic Re~Qn~nce Imaaina ..
; ~ In vi~o magnetio resonance imaging of human organs and tissue is conventional and weli established.
~igure 5 is a photograph of T-l we~ghted magnetic resonance images o~ a rat obtained before, and at 0, 5, 10, 15, 25, 45, and 60 minutes after the injection of Gd-DTPA-bi~(phenylalanine) at a dose of 0.1 mmol/kg body weight.
The images are 60 mm x 60 mm x 3 mm thick slices in th~
:: SU~STITUlE SHEET
IPEA/US

16 Re~'d PCTlPT0 0 ~ 993 rT/us 92 /06660 2 ~ 7 ~

coronal plane. The region covered extends from just above the heart to somewhat below the liver. Enlargements of the pre- and O-min post images are shown in Figure 5B. Imaging parameters are indicated along the left of ths Figure and include the repetition time (3000000 microseconds), echo time (ÇOOO microseconds, number of signal averages (43, and the image matrix size (128 x 256). The increase in signal intensity, particularly in the heart and liver, are readily apparent. Increas2 in signal intensity of the intestinal lumen is particularly apparent in the 25 min and later images, and suggests that contrast agent has been excreted into that organ.
Figure 6A and 6B are photographs of T-l weighted magnetic resonance images obtained as described in Fig. 5A
and 5B, except that Gd-DTPA-bis(phenylalanine ethyl ester) was used as the contrast agent. Note that this compound results in different apparent enhancement in the liver and heart as compared to that shown in Fig. 5A and 5B. These results suggest that the two compounds have significantly different biodistributions and pharmacokinetics.
; Specific experiments are described in detail below in the Examples.
Administration of Contrast Aaent ~ ~ Any physician can determine the best mode of -~ 25 administration o~ the contrast agent. Generally, injection into a vein is used.
The contrast agents described herein are useful for the magnetic resonance imaging of the heart, liver, biliary ~ tree, bladder and intestine of a subject, e.g. an animal, a mammal, especially a human being.
The following Examples are provided to further explain and describe the present invention. They are not to be construed to be limiting in any way.
' ~ 35 E%AMPLE 1 SU~TIl UrE SHEEr ,, IPEAIUS

16 R~'d PCTIPTO O 9 MI~R 1993 ~CT/ U S 92 ~ 0 66~0 18 21 1~27~
PREPARATION OF DTPA-BISlPHENYLG~Y~INE~
In a 50-mL r~und-bottom flask equipped with a magnetic stirrer and a reflux condenser, and heated by an oil bath, was placed 1.10 g (3.08 mmol) of DTPA-bis(anhydride) (Aldrich Chemical Co.), 0.93. g (6.}5 mmol) of d,l~
phenylglycine tFluka Chemical Co.), and 25 mL of dry dimethylformamide (Aldrich Chemical Co.). The reaction mixture was heated to 90-100~ and held within that temperature range for 6 hr. It was then allowed to cool to room temperature~ and the solvent w~s removed using a rotary evaporator. The residue was washed by trituration - with ether to yield 2 g of white solid of structure Ib (Figure 3).
EXAMPLE_~
15PREPARAT~ON OF THE Gd(III~ COMPL~XES
OF DTPA-BIS(PHENYL5~5~El ~ solution of 2 mg (30 ~mol) of DTPA-bis(phenyl-glycine) of Example 1 in 1 mL of water was treated with 14 20mg (38 ~mol) of GdCl3-6H2O. The pH of the resulting w~s adjusted to 7.0 by addition of dilute sodium hydroxide solution. Insoluble Gd(OH) 3 was removed from the reaction mixture by filtration through a 0.22 ~ filter. The T1 : relaxation time of the resulting solution (1.3 mL Yolume) 25was 7 millisecond (ms)) at 0.25 Tesla and 37C.

MA~NETIC RESONANCE IMAGING OF A RAT_USING
Gd-DTPA-BISfPHENYLGLYCINE) 30A 300 g male Sprague-Dawley rat was anesthetized with a intraperitoneal injection of a mixtuxe of ketamine and ~ -diazepam, and a catheter was inserted into a lateral tail Yein. The rat then was placed in a 5-cm inside diameter (i.d.) imaging coil in the bore of a 2-Tesla imager-8pectrometer system (GE CSI; General Electric Co., Fremont, California). A Tl-weighted spin-echo ima~e of the animal's abdomen in the coronal plane was then o~tainad (TR 315 ms;
~UBSTITUrE SHEET
, IPEA~US

-!~ l6R~c'dPCT/PTO 09MAR1993 `
E"`T/ U S 92 / 0 6660 19 211ia27~
Te 15 ms; 128 x 256 image matrix; NEX = 4; 3 mm slice thickness). Next, 1.0 g of the Gd-DTPA-bis(phenylglycine) solution described in Example 2 was injected via the catheter~ A series of post-inje~tion images were obtained.
The images displayed an initial small enhancement in the liver. As this enhancement decreased with time, increased intensity in the rat's small intestine then was observed, indicating hepatobiliary transport of the contrast agent.
Intensity data are summarized below.
:~ 10 TABLE 1 IMAGE REGION-OF-INTEREST % EN~ANCEMENT
Time Liver Small In~estine Musc~e (min post injection) ~: ~ 15-18 7 42 2 ~:~ 30-33 2 32 6 X~MPLE 4 ~: PREPARATION.OF DTPA-BISfL-PHE~YALANINE ETHY~ ESTER) L-phenylalanine ethyl ester hydrochloride, 4.6 g (20 mmol, Sigma Chemical Co., St. Louis, Mo), was dissolved in ~: 15 mL of water and treated with 35 mL of saturated sodium bicarbonate solution. The resulting solution was extracted ~, , . with four 10 mL portions of methylene chloride, and the ~ 30 organic extract was dried over anhydrous magnesium su}fate.
; The dried methylene chloride solution then was filtered to remove remaining drying agent, and the filtrate was concentrated to an oil using a rotary flash evaporator.
This residue was further dried under high vacuum for -35 several hours to yield 3.~5 g of free base. ~~~~
DTPA-bis(anhydride~, 2.85 g (8.0 mmol), 10 mL o~ di-methylformamide ~DMF), and 4.2 m~ (24 mmol) of diisopropyl-ethylamine (DIPEA) (Sigma Chemical Co., St. Louis, M0) w~re combined in a 50 mL round-bottom flask eq~ipped wlth a - SUBS;~ ET

t6 R0c'd PCT/PTO O 9 MAR l993 PCT/I~ 92 ~0 6660 ~ 1527~
magnetic stirrer. The phenylalanine described above was dissolved in lO mL of DMF, and the resulting solution added ~ia syringe to the flask. The reaction mixture was warmed to 40C, and then stirred for 13 hr at ambient temperature without external heating.
At th~ end of the 12 hr period, the reaction mixture was concentrated in vacuo to yield a viscous residue. This material was triturated with lOO mL of acetone, and the volatil~ components of the r~sulting ~ixture were removed in vacuo. The solid residue was recrystallized ~ro~ a mixture of 125 mL of 60/40 water/~thanol. The white, crystalline product was washed with two 25mL portions of cold ethanol, and the washed solid was dried in vacuo at 40C for l hr to obtain 3.0 g (50% of theory).
Analytically pure product was obtained by dissolving 1 g of the above crystals in 75 mL of ethanol at 80-~5C, treating the resulting solution with decolorizing charcoal, removing the latter by filtration, and cooling the filtrate in an ice bath. Seed crystals were then added, and after 20 45 min, 0. 6 g of recrystallized solid was isolated by filtration.
Anal: Calcd. for C36H~50~2: C, 58.13; H, 6.64; and N, ~: 9.42. Found: C, 57.75; H, 6.57; and N, 9.3S.

PREPARATION OF DTPA-BIS(L-PHENYLALANINE ~ENZYL ESTERL
DTPA-bis(phenylalanine benzyl ester~ was similarly prepar2d (according to Example 4) from L-phenylalanins benzyl ester p-toluene-sulfonic acid salt, 4.28 g (10 ~mol;
Sigma-Chemical Co., St. Louis, MO). Ethyl ac~tate was us¢d in place of ethanol for recrystalli7ation. Th~ yield was 2.6 g (75% of theory).

PREPARATIO~ OF DTPA-BIStL-PHENYL~L~NINE) A solution of 1.23 g (1.42 mmol) o~ DTPA-bis(phenyl-alanine benzyl ester) in ~5 mL of methanol was combined SUBST'TUTE S~
, IPEkJ~J

16 ~ec~d ~GT/P~O O 9 ~AR 1993 ~TI U~ 92 / 0 6660 211~27~

with 0.1 g Pd/carbon catalyst (Aldrich Chemical Co,Milwaukee, WI) in a 2S-mL round bottom flask. This mixture was treated with hydrogen gas at one atmosphere pressure for 6 hours. The reaction mixture was then filtered through a bed of diatomaceous earth filter aid. Volatile oomponents were removed from the filtrate in vacuo. ~he yield was 0.94 g (97% of theory) of product, a somewhat hygroscopic white solid.
Anal: Calo'd. ~or ~32H3~N5O~2*2HOH: C, 53.10 H, 6.27; and 10 N, 9.68. Found: C, 53.13; H, 6.24; and ~, 9.32.
When examined by HPLC, see description for Figure 8 below, the product was found to be about 10% bis acid, 45%
bis ester and 45% mono acid mono ester. This-is actually the contrast agent used for the Figure 6 MRI image.
EXAMPLE ?
PREPARATIO~ OF THE GADOLINIUM (III) CHELATEs OF
BIS(p~ENYLALANINE) AND I~S ~STERS
(a) A solution of 0.176 g (0.25 mmol) of DTPA-bis-(phenylalanine) in 4 mL of water was treated with 0.093 g of GdCl3 (Aldrich Chemical Co., Milwaukee, WI). The pH of the resulting solution adjusted to 7.0 with aqueous sodium hydroxide solution. The vol~me was adjusted to 5.0 mL with water, and this solution was filtered through a 0.2~ micron sterile filtex into a sterile serum vial. The resulting : 0.05 M solution i suitable for imaging in small animals.
The T~ relaxation time at 0.25 Tesla magnetic field strength and 37C of a five-fold dilution of the above solution was 21 ms.
,.
3Q~ tb~ The DTPA-bistphenylalanine) mono and bis esters were prepared in a similar ~ashion.
EXAMPLE 8 ~~~~
~GNETIC RESONANCE IMAGING OF A RAT U$ING
Gd-~TPA-BIS~PHENYLALANINE) A 300 g male Sprague-Dawley rat was an~sthetized with a intraperi on~al injection of a mixture of ketamine and SUBSTITUTE SHEET
IPEA~US

16 Rec'd ~GTlP~O O 9 ~R lS, ~T/US 9~06~60 22 2i 1~27~
diazepam, and a catheter was inserted into a lateral tail vein. The rat then was placed in a 5-cm inside diameter (i.d.) imaging coil in the bore of a 2-Tesla imager-spectrometer system (GE CSI; General Electric Co., Fremont, California). A Tl-weighted spin-echo image of the animal's abdomen in the coronal plane was then obtained (TR 300 m ;
Te 6 ms; 128 x 256 image matrix; NEX - 4; 3 mm slice thickness). Next, 0.6 g of the Gd-DTPA-~is(phenylalanine) solution described in Example 7 was injected via the catheter. A seri s o~ post-injection images were obtained.
The i~ages displayed an initial enhancement in the liver and heart. As this enhan~ement decreased somewhat with time, increased intensity in the rat's small intestine then was observed, indicating hepatobiliary transport of ~he contrast agent. Intensity data are summarized below. The - intensity values show some fluctuations due to breathing motion and other small artifacts.
TABLE_~
IMAGE REGION-OF-INTEREST % ENHANCEMENT
Time Liver Heart Muscle (min post injection~
: 0-3 51 42 14 ~-8 60 27 3 25-3~ 50 22 12 . 45-48 34 9 10 3~

MAGNETIC RESONANCE IMAGING OF A RAT USING ~ ~
Gd-DTPA-BIS(PHENYLALANI~E ETHYL ~STER~
The imaging was carried out analogously to Example 8.
Intensity data are summarized below.

STITUTE Sl I~Er IP.'~

`` 16 Rec'~ ~CT/PTO O 9 MAR 1993 ~T/us~922~o66B

IMAGE REGION-OF-INTEREST % ENH~NCEMENT
Skeletal Time Liver Heart Muscle (min poct injection) 0-3 7~ 86 50 15-~8 113 66 31 60-~3 44 25 16 (BIS PHE_ACID ABOUT 100%1 FIGURE_7 Two male BALB mice were imaged side-by-side in the same apparatus and using.th~ same conditions as found in Example 8, except that the slic~ thickness was 2 mm. Over the illustrated time course from 0 min to 2.5 hr, th~
contrast agent can be seen to localize f irst in the liver (e.g. at 2 min), then in the gall bladder (at 90 min, for example~, and then in the intestinal lumen (2-2~5 hr). It can also be seen in the urinary bladder.

COMPARATIVE MRI pATA_IN MICE
: ~
Figure 8 is a photograph of T-l weighted magnetic resonance i~ages obtained as in Figure 6, except that a different pr~paration of Gd-D~PA-(Phe-Et32 was used.
When examined by HPLC (4.6 x 150 mm PRP-1) column;
mobile..phase -25 mmolar a~monia formate in water (Solvant ~ A) and 50/50 (U/V) acetonitrile/water (Solvent B), programmed from 10% B to g5% B over 15 min, then holding at 95% B; flow rate 1 ml/min; U/Y and/or radioisotope detector, the material used as a contrast agent in Figure 6 was found to ha~e partially hydrolyzed to a mixture of Gd-DTPA- (ca 45%), Gd-DrPA-~he-Et~ (Phe) (ca 45%), and Gd-SUBSTITUTE SHEET
IPEhl~)S

t6 R~'d ~T/PT0 0 9 MAR 199' ~./US 92/U~660 24 ~11527 DTPA-(Phe)2(Ca 10%)-Freshly prepared material, whose pH was carefully adjusted to neutrality, and which was st~red in the cold, was determined to be about 90%. Gd-DTPA-~Phe-Et~ the remainder being mostly Gd-DT~A-(Phe)(Phe-Et~.
The more pure preparation gave heart and liver enhancement (74 and 163% resp.) as shown in Figure 6 (84 and 53%, resp.). Thus the degree of liver enhancement was greater by a factor of about three.
io These results suggest that esterified DTPA-amino acid chelates may be particularly advanta~eous for lower contrast enhancement. - ~:
Example 12 Preparation of DTPA-~is(D-Phenylalanine Ethyl Ester~
DTPA-Bis(D-Phenylalanine Ethyl Ester) was prepared analogously to the rorresponding L-isomer from D-phenylalanine ethyl ester and DTPA-bis(anhydride) (Example 4). The yield was 65%.
Anal. Calcd for C3~H49NSol2: C, 58.13;H, 6.64; and N, 9.42. Found: C,57.87; H,6.55; N,9.48.
Example 13 Preparation of DTPA-Bis(Phenylalanine Methylamide~
A suspension of 2.07 g ~10.43 mmol) of L-phenylalanine methyl amide hydrochloride in ethyl acetat~ (75 mL) was treated with a saturated aqueous solution of sodium carbonate (20 mL~. The resulting solution was extracted with ethyl acetate (2 x 75 mL), and the combined organic ; extracts were dried over anhydrous sodium sulfa~e. The drying agen~ was removed by filtration, and the filtrate 30 was conrentrated to an oil using a rot~ry evaporator. This residue was further dried over P20S under high vacuum -~~~
overnight to yield 1.72 g of ths amine as a white solid.
A solu~ion of the dried amine in anhydrous pyridine (15 mL) was comb~ned with DTPA-bis(anhydride) (1.80 g, 5.04 mmol)under argon. Tbe reaction mixture was heated at SU~STITUrE SHEET
~PEA/IJS

16 Rec'd PC~/PT0 0 9 Y;AR 199~

211~ 2 7 5 reflux in An oil bath (95 C) for 60 min. The mixture was allowed to cool to room temperature ~0.5 hr) and was concentrated in vacuo to yield a viscous residue. This material was dissolved in 100 mL of water, and the water then was evaporated in vacuo to yield a yellow oil. The oil was dissolved in a minimum amount of a solution of water in methanol (20% v/v) and treated with acetonitrile until a small amount of precipitate was observed. The precipitate was xemoved by filtration (0.45 ~m membrane filter), and the filtrate was concentrated under reduced pressure. This procedure was repeated twice more, discardi~g the precipitate each time. Finally, the residue obtained by evaporation o~ the solvent was dissolved in a solution of water in methanol (lo mL, 20% v/v), and the desired product was precipitated by addition of a minimum amount of - acetonitrile. ~he resulting whita suspension was cooled in a freezer (-20- C) overnight, and the solvent then was removed by decantation. The product was dried under high vacuum (O.Q5 torr, 48 hr~ over P20~ ~nd NaOH to afford 1.38 g (39%) of an analytically pure white solid.
Anal. Calcd. forC~H47N~0l0~.5 H20:C, 56.50; H, 6.69;
N, 13.57. Found: C, 56.34; H, 6.61; N, 13.66.
:
Exam~le 14 P~eparation of DTPA-Bis(Phenylalanine Amid~Land ~TPA-~25 BisrPhenylalanine Dimethylamide~
The title compounds were prepared analogously to th~ ~-dimeth~lamide compound (Example 13) from DTPA-bis(anhydride) and L-phenylalanine amide hydrochloride and L-phenylalanine dimethylamide in %20 and %59 yields, ~ , respectively.
Calcd for the amide ~ H~3N7~o-2H2o C~53-25~ H~6-57;
N,13.58. Found: C,53.43; H,6.30; N,13.59.
Calcd for the dimethylamide C~H5lN~010 C~56-90;H~7-03;
and N,12.90. Found: C,56.82;H,6.74;N,12.76.
Example 15 SUBSnTUTE SHE~T
IPEAIU~

` 16Rgc'dPCTtPTO 09MAI~1993 :
/U~ 6~6Q
26 2 1 1 ~ 2 7 ~
Preparation of DTPA-bis(3-hydroxytyramide~
~"DTPA- ! 3-HTAl,~
DTPA-bis(anhydride) (3.57 g; }.00 mmol; Aldrich Chemical Co., Milwaukee, WI) was suspended in 25 mL of anhydrous dimethylformamide (DMF; Aldrich) and treated with dopamine (3.78 g; 2.00 mmol; Fluka-USA, Ronkonkoma, NY) and di-isopropylethylamine (5.2 g; 4.0 mmol; Aldrich Chemical Co.) This mixtu~e then was heated briefly to lOO C and ~onicated for several mi~nutes to dissolve the bulk of tha solid. After tirring for 4-~ hr at 50-60-C, a d~ep yellow solution was produced. The reaction mixture then was allowed to cool to room temperature.
After stirring at ambient temperature overnight, the reaction mixture was concentrated on a rotary evaporator at 50 C to a volume of about lo mL. The odor of di-isopropylethylamine was absent at this point. water (25 mL) was added, and the resulting -~olution was washed twice with 20 mL portions of ethyl ether to remove the remaining DMF. The water then was removed in V3CU0 to yield a beige paste. This material was suspended in 10-20 mL of absolute ethanol and dried by azeotropic distillation of aqueous ethanol in vacuo. The crude product (7 g; 97~ of theory) was a gritty, off-white, hygroscopic solid.
An analytical pure sample (1.76 g; 26% of th ory) was isolated by preparative high-pressure liquid chromatography (HPLC) using a 4.6 x 150 mm Microsorb C-18 reversed phase column (Rainin Instrument Co., Emeryville CA). The mobile phase (1 mL/min~flow rate~ was a linear gradi~nt from 5 to 50% acetonitrile in water over 12 min. An acidic p~ was maintained by the presence of 0.1% v/v trifluoroacetic acid in both compon~nts of the mobiie pbase. A W detector measuring ab~orbance at 276 nm was u~ed. The retention time o~ the product was 13.5 min under these conditions.
JH NMR spgctrum: ~ 6.75, m, 6H; ~ 3.81-2.62, 26 H, aliphatic H, not ~urther assigned.
SUBSTITUTE SHEET `
'' IPEA/IJS

16 Rec~d PCTlPT0 0 9 MAR l993 l U S 9 2 1 0 6660 27 211~27~
Liquid secondary ion mass spectrum (LSIMS) tM-H]- = 662 (theory 662).
Example 16 Preparation of _DT~A-bis(3.4-dimethoxvphenethYla ide) ("DTPA ~4-DMPE)~
~uimolar amounts of DTPA-bis(anhydride) and 3,4-dimethoxyphenethylamine (Aldrich Chemical Co.) were contacted as above in Example 15 to give crude product in ca 100~ yield. This material was purified by preparative 10 HPLC to produce 1.23 g (17%) of the title compound.IH NMR spectrum: ~ 6.80, m, 6H; S 3.82, s, 6~I, CH30-; ~ 3.80, s, 6H; CH30-; ~ 3.45-2.76-, 26 H, aliphatic H, not further assigned.
LSIMS mass spectrum: ~M-H~- = 718 (theory 718).
Example.17 Preparation of Gd-DTP~-(3-HT~12 Solutions of Gd-DTPA-(3-HTA) 2 for imaging exp~riments were prepared by reacting DTPA-(3-HTA~ 2 in aqueous solution with a stoichiometric amount o GdCl3 dissolved in water. After about 90% of the GdCl3 had been added, the pH of the reaction mixture was adjusted to between 5 - and 6 with aqueous NaOH solution. Xylenol orange indicator (1 drop of a 1 mg/mL aqueous solution) then was added, and GdCl3 solution was added dropwise until the ~ 25 indicator changed from yellow to violet (at pH <6). The -~ pH then was adjusted to between 7 and 8 with aqueous NaOH
and, if necessary, aqueous Hcl solution. The reaction mixture was passed-through a 0.22 ~m sterile filter into a sterile serum vial. The final concentration ranged ~: 30 from 0.02 to 0.5 M, depending upon the initial concentrations of the reactants and the volumes of base ~~
and acid added for pH adjustment.
A sample o~ product for mass spectral analysis was obtained by HPLC (4.6 X 150 PRP-l column; 1 mL/min flow rate; 5-45% over 15 min aceton~trile-in-water gradient).
SU~sSTITlJ ~Tt ~HEE~I
IP~8 ., 16 Rec'~ P~T/PTO O 9 MAR 1993 28 ~ 11 a2 7 ~
The LSIMS [M+H]+ parent ion peaks were observed from 815-824, with the maximum intensity at 819. The ratios of peak intensities were those predicted by theory C3oH3~GdN5ol2 -Exam~le 18 ~re~aration of Gd-DTPA-~3,4-DMPE~
This chelate was prepared analogously to Gd-DTPA-(3-HTA)2 Example 17, above ~rom DTPA-(3,4-DMPE) 2 and GdCl3 Exam~le 19 In Vivo Maqnetic Resona~ce I~a~inq Usin~
~d~Tp~-~3-~TA)~.ands~ 2~:LL-L~ L2 Magnetic resonance imaging was carried out using a CSI 2 Tesla imager ~GE, In~., Fremont, CA) equipped with a 5-cm diameter distributed-capacitance imaging coil.
T1-wei~hted (TR 300/TE 6; NEX 4) spin-echo se~uence was used. The image matrix was 128 X 256, the slice thickness was 3 mm, and the field-of view was 90 mm.
Anterior (heart level) and posterior (kidney level) coronal image planes were used.
Sprague-Dawley rats (250-350 g; n = 4 for each contrast ~gent) were anesthetized with ketamine (90 :- mg/kg) and diazepam (10 mg/kg) and fitted with an intravenous catheter in a lateral ~ail vein. Anes~hesia a was maintained during imaging using pentobarbital delivered viz an intraperitoneal catheter.
: The anesthetized animal was placed in the imaging coil and secured with tape. The coil containing ths animal then was placed in the magnet bore, and the magnetic field was shimmed. Pre-contrast i~ages were obtained. The contrast agent (100 ~mol/kg) ~hen was injected via the tail-vein catheter, and additional -~ ~
images wer~ obtained at various interval~ for up to 90.
~in post injection.
Contrast agent enhancement was determined by measuring the mean signal intensity (SI) in operator-IPEAllJS

16 Rec'd PCTIPTO O 9 MAR 19~3 P~T/ US 92 /06~;60 29 211a27~
designated regions of interest (ROI). These were normalized to the pre-injection value for each ROI
according ~o the following formula:
% Enhancement - 100 X (SIpost - SIpre)lSIpre The contrast enhancement (mean + s.d., n = 4~ as a function of time in heart, lung, kidney, liver, and skeletal muscle, Figures 9 - 13 respectiYely, for each of the contrast agents are illustrated, Gd-DTPA-(3-HTA)~also tended to produce hiqher lung enhancement ~186% + 51% vs.
141% + 4%). However, the differences between the effects produced by the two agents was smaller than in heart ~cf.
~igs. 9 and 10).
There was no significant difference in kidney enhancement (Fig. 11). Both agents produced ca. 175%
enhancement 5 min after injection. The level of enhancement fell slowly over 70 min to about ~00%.
About 50% enhancement was produced in the liver by both agents immediately post-injsction (Fig.l~).
Additionally, the time course of enhancement was very similar for both agents, with the enhancement level falling to about 30% during the first 20 min post ` injection.
Skeletal muscle displayed peak enhancement of about ; ~ 40~ immediately post-injection. The enhancement-time 25 curYes for both agents were almos~ iden~ical; each fell almost to pre-injection levels over 80 min (Fig.13).
Representative images using each agent are shown in Figures ~4A and 14B and 15A and 15B as MRI photographic images.
~0 Example 20 In Vivo Maanetic Resonance.Ima~ina Usina_ Gd-DTPA-(L-PheOEt)2 and Gd-DTP~D-PheOEt~ 2 The magnetic resonance imaging characteristics of the two contrast agents Gd-DTPA-(L-PheOEt) 2 and Gd-DTPA-(D-PheOEt)2were compared as in the pr~vious' Example using SUBSTITI~E SHEET
'' IPEA~S

16 Rec'd PC~/PTO O 9 M~R t9~3 - P~T/US 92~0~660 30 ~ 7 ~
groups of 4 and 5 animals, respectively. Figures 16 to 20 illustrate the ~ontrast enhancemPnt ~ersus time behavior for each agent in heart, lung, kidney, liver and skeletal muscle, repectively.
Representative images using each agent are shown in Figures 21A and 21B and 22A and 22B as MRI photographic images.
Exam~le 21 HYdrolysis of Gd-DTPA-fL-PheOEt)2_3n~_ Gd-E~ in pH.7.4 ~uf~er ~n~ ~at ~l~a~
Th~ rates of hydrolysis of th~ est~rs in rat plasma or pH 7.4 HEPES buffer were detsrmined by addition o~
10% by volume of Gd-153 radiolabeled 0.025 M d elate solution and incubation at 0 or 25 C. Aliquots were withdrawn at various time intervals and examined by HPLC
~PRP~1 column; water-acetonitrile gradient; 25 Mm ammonium formate, pH 7 mobile phase3.
: Th~ hydrolysis of either the ~L- or DD-bis(ester~
enantiomers to th~ corresponding mono(ester)-~ono~acid3 ~: 2~ and thence to the bis(acid) in aqueous HEPES buffer at pH
: 7.4 and 25 C is very slow, with half-times for each step : of the order of days.
However, the LL-bis(ester) is very rapidly :~ hydrolyzed in rat plasma to the mono(acid)-mono(ester) (see below). The latter compound is much more resi tant to hydrolysis of the remaining ester, with essentially no reacti.on being observed within 2 hr at 25 C.
In contrast, th~ DD-bis(ester) is resistant t~ even the ~irst step of ester hydrolysis under the~e conditions 3 ~ (see below).
: tln_of Ester Hydro~ ~$ in_Rat Pla5ma.
0-C ~ 25-~
Gd-~TPA-~L-PheOEt32 33 min 0.3 min Gd-DTPA-(D-PheOEt)2 None Detected None Detected ~LiBSTlTu~E SH~
IP~ S

16 R~c'd PCTlPTO O 9 MAR lS9~:
` PCT/-US 92/06660 31 211~27~
The relative stability of the bis(esters) toward hydrolysis in aqueous solution versus plasma suggest that the plasma reaction is enzyme-catalyzed. Furthermore, mono(acid)-mono( ster) is evidently a much poorer sub trate, as its rate of hydrolysis is much slower.
This may be due to the change in net charge ~from O to -1) of the chelate and/or to a change in conformation of the molecule due to coordination of the Gd by the free phenylalanine rarboxylate group.
Changing the stereochemistry of the amino acid portion of the chelat~ to the unnatural D-enantiom@r caused the rate of ester hydrolysis in plasma to greatly decrease.
ExamPle 22 Determination of Relative AmouQts of Urinary and Biliary Excretion Male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of mixture o~ ketamine (90 mg/kg~ and diazepam (2 mg/kg), and fitted with a 23-guage 20 cannula placed in a lateral tail vein. Next, a midline inci~ion and a small lateral cut over the bile duct were made, and the bile duct was exposed. Two loose ties were pla~ed proximally on the bile duct. A small nick was made distally, and the bile duct was cannylated with a , 25 15 cm leng~h of PE-10 polyethylene tubing, which wa~
secured with the two ties.
A second piece of tubing was placed in the urinary bladder and secured with a purse-string suture. The flap of the abdominal wall was closed, and the incision was 3~` -covered with gauze.
Heparinized ~1 unit/mL) saline was infused at a rate of 0.075 mL/min via the iv catheter. A~ter a 15 min stabilization period, the infusion was interrupted lony enough to deliver a bolus dose tO.l mmol/kg) o~ Gd-153 labeled contrast agent, and then resumed. Samples of SUBSTlTUrE SHEET

16 Rec'd P~ O 9 MAR 1993 .
' 3 PCT/Us 92~06660 3~ ~la.;~7~
bile and urine were collected in tared tubes at regular intervals before and after injection of radiolabeled agent. The net weights of these samples were deter~ined.
The amount of Gd-153 present in each sample was 5 determined by counting in a chamber gamma counter. The raw counts were corrected for background and normalized to the total amount of Gd-153 injected.
The Table below summarizes the results (cumulative 1 hr excretion; average of 3 animals~ obtained for some of 10 the agents described in the prior Examples:
One-Hour Cumulative ~xc e~ion BiliarY . U~inar~
Gd-DTPA-(L-Phe~2 9.3+1.3 66.5+8.7 Gd-DTPA-(L-PheOEt) 2 30~5+7 4 46.g+8.0 Gd-DTPA-(D-PheOEt) 2 51.3+5.1 39.2+5.5 Gd-DTPA-(L-PheNHCH3)2 3 5+0 4 70.9+6.5 While only a few embodiments of the invention have been shown and described herein, it will become apparent to those skilled in the art that various modifications and changes can be made in the amino acid containing hepatobiliary or cardiac contrast agents or their use in magnetic resonance imaging of the torso or abdomen of a mammal without departing from the spirit and scope of the P 25 present invention. R or Rl groups of the ligand L
optionally comprise an aromatic or heteroaromatic moiety.
All such modifications and changes coming within the scope of the appended claims are intended to be carried out thereby.
_._ .~ JBST~TU-~ S~
~p~BJ~

Claims (42)

WE CLAIM:

1. A magnetic resonance imaging contrast agent, comprising the complex:
L-M
wherein M is a metal (II) or (III) ion independently selected from the group consisting of metals of atomic number 21 to 31, the lanthanide metals having an atomic number from 57 to 82, and L is a polydentate organic chelating moiety of structure Ia:

(Ia) wherein Q, J, Y and Z and X and X1 when present are each independently selected from -CH2(C=O)-OH, or -CH2(C=O)NHCH-(R)-(A);
wherein R in each of Q, J, X, X', Y and Z is independently selected from hydrogen or an organic structure comprising an alkyl, aryl, substituted aryl, alkylene aryl, alkylene substituted aryl, heteroaromatic, substituted heteroaromatic, alkylene heteroaromatic, or alkylene substituted heteroaromatic group provided that at least one of Q, J, Y and Z is -CH2(C=O)-NHCH(R)-A; and A is independently selected from -(C=O)OR1, -(C=O) -N-(R2)R3, or R4 wherein R4 is independently selected from -CH2-aryl, -CN2-substituted aryl, -CH2CH2-aryl, or -CH2CH2 substituted aryl, provided that when A is - (C=O) OR1 and R1 is hydrogen then R is not hydrogen;
R1, R2 and R3 when present in A in each of Q, J, X, X', Y and Z are independently selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl; and m is selected from 0, 1, 2 or 3, and n is selected from 0 or 1, or the pharmaceutically acceptable salt(s) thereof.

2. The contrast agent of Claim 1 wherein the .alpha.-amino acid(s) when present are selected from the D or L
configuration or mixtures thereof.

3. The contrast agent of Claim 2 wherein at least one of J or Z is -CH2(C=O)NHCH-(R)-(A) and A is -(C=O)-N(R2)-R3, and R2 and R3 are independently selected from H
or alkyl having 1 to 6 carbon atoms.

4. The contrast agent of Claim 3 wherein Q and Y
are -CH2(C-O)-OH and X, X1 when present are -CH2(C=O)-OH
and m is O or 1 and n is O.

5. The contrast agent of Claim 4 wherein one of R2 or R3 is hydrogen.

6. The contrast agent of Claim 5 wherein both J
and Z are each -CH2(C=O)NH(CH)-R(A).

7. The contrast agent of Claim 4 wherein R2 and R3 are each alkyl.

8. The contrast agent of Claim 7 wherein J and Z
are each -CH2(C=O)NHCH-(R)-(A).

9. The contrast agent of Claim 1 with the priviso that at least one R or A comprises an aryl, substituted aryl, alkylenearyl, alkylene substituted aryl, heteroaromatic, substituted heteroaromatic, alkylene heteroaromatic, or alkylene substituted heteroaromatic group.

10. The contrast agent of Claim 1 wherein Q, X, X' and Y are each -CH2(C=O)-OH, and at least one of J and Z
are -CH2(C=O)NH CH(R)-A wherein A is -CH2-aryl, -CH2-substitut-ed aryl, -CH2CH2-aryl, or -CH2CH2-substituted aryl, wherein aryl is selected from phenyl or naphthyl, and substituted aryl is substituted with 1 to 3 groups independently selected from halogen, alkyl having 1 to 7 carbon atoms, hydroxy, alkoxy having 1 to 7 carbon atoms, nitro, nitro-so, amino or trifluoromethyl.

11. The contrast agent of Claim 10 wherein A is -CH2- substituted aryl wherein aryl is phenyl and is sub-stituted with 1 to 3 alkoxy or hydroxy groups.

12. The contrast agent of Claim 11 wherein m is 0 or 1, n is 0 or 1; Q, X, X1 and Y when present are each -CH2(C=O)-OH, and at least one of J and Z are -CH2(C=O)NH-CH(R)-A, wherein R is hydrogen and A is -CH2-substituted aryl and is substituted with two alkoxy groups.

13. The contrast agent of Claim 12 wherein J and Z
are -CH2(C=O)NH(R)-A.

14. The contrast agent of Claim 11 wherein A is -CH2- substituted aryl wherein aryl is phenyl and is sub-stituted with two methoxy groups.

15. The contrast agent of Claim 12 wherein m is 0 or 1, n is 0 or 1; Q, X, X1 and Y when present are each -CH2(C=O)-OH, and at least one of J and Z are -CH2(C=O)NH-CH(R)-A, wherein R is hydrogen and A is -CH2-aryl and is substituted with two hydroxy groups.

16. The contrast agent of Claim 15 wherein J and Z
are each -CH2(C=O)NH(R)-A.

17. The contrast agent of Claim 11 wherein m is 0 or 1, n is 0 or 1; Q, X, X1 and Y when present are each -CH2(C=O)-OH, and at least one of J and Z are -CH2(C=O)NH-CH(R)-A, wherein R is hydrogen and A is -CH2-aryl, aryl is phenyl and is substituted with two hydroxy groups at the 3 and 4 positions of the ring, or with two methoxy groups at the 3 and 4 positions of the ring.

18. A magnetic resonance imaging contrast agent, comprising the complex:
L-M
wherein M is a metal (II) or (III) ion independently selected from the group consisting of metals of atomic.
number 21 to 31, metals of atomic number 39 to 50, the lanthanide metals having an atomic number from 57 to 71, and metals of atomic number 72 to 82, and L is a polydentate organic chelating moiety of str-ucture Ia:

(Ia) wherein Q, J, X, X', Y and Z are each independently selected from -CH2(C=O)-OH, or -CH2(C=O)NHCH-(R)-A, wherein A is COOR1; R in each of Q, J, X, X', Y and Z is independently selected from hydrogen or an organic structure comprising an alkyl, aryl, alkylene aryl, alkylene substituted aryl, heteroaromatic, alkylene heteroaromatic group, substitut-ed heteroaromatic, or alkylene-substituted heteroaromat-ic, provided that at least one of Q, J, Y and Z is -CH2(C=O)-NHCH(R)COOR1 provided that when R1 is hydrogen, then R is not hydrogen, R1 in each of Q, J, X, X', Y and Z is independently selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl; and m is selected from 0, 1, 2 or 3, and n is selected from 0 or 1, or the pharmaceutically acceptable salt(s) thereof.

19. The contrast agent of Claim 18 wherein the .alpha.-amino acid(s) is selected from the D or L configuration or a mixture thereof.

20. The contrast agent of Claim 19 wherein M is a paramagnetic metal ion (II) or (III).

21. The contrast agent of Claim 20 wherein Q and Y
are each -CH2(C=O)NHCH(R)COOR1, J, X, X1, and Z are each -CH2(C=O)OH, and m and n are each 0.

22. The contrast agent of Claim 21 wherein R is -CH2-phenyl, and R1 is hydrogen.

23. The contrast agent of Claim 18 wherein M is a (III) metal ion, and Q and Y are each -CH2-(C=O)NHCH(R)-COOH wherein R is selected from benzyl, p-hydroxyphenylmethyl, 2-methyl-naphthyl, or 3-methylindolyl;
J and Z are each -CH2(C=O)-OH, m is 0 or 1, and X is - CH2(C=O)OH, and n is O.

24. The contrast agent of Claim 23 wherein the metal ion is selected from chromium (III), iron (III), cobalt (III), gadolinium (III) or manganese (II) or (III).

25. The contrast agent L-M of Claim 18 selected from the group of compounds of structure I consisting of the following groups:
M is independently selected from iron (III), chromi-um (III), manganese (II), dysprosium (III) or gadolinium (III);
Q is -CH2-(C=O)NHCH(R)-COOH, R is -CH2-phenyl, J, Y
and Z are each -CH2(C=O)OH, and m and n are each O;
Q is -CH2-(C=O)NHCH(R)-COOH, R is -CH2-phenyl, J, X, Y and Z are each -CH2(C=O)OH, and m is 1 and n is O;
Q is -CH2-(C=O)NHCH(R)-COOH, R is -CH2-phenyl, J, X, X', Y and Z are each -CH2(C=O)OH, and m and n are each 1;
Q and Y are each -CH2-(C=O)NH-CH-(R)-COOH, R is -CH2-phenyl, J and Z are each -CH2COOH, and m and n are each O;
and Q and Y are each -CH2(C=O)-NH-CH-(R)-COOH, R is -CH2-phenyl, J, X and Z are each -CH2COOH, and m is 1 and n is O.

26. The contrast agent of Claim 18 with the priviso that at least one R or one A comprises an aryl, substi-tuted aryl, alkylenearyl, alkylene substituted aryl, heteroaromatic, substituted heteroaromatic, alkylene heteroaromatic, or alkylene substituted heteroaromatic group.

27. A composition useful as a contrast agent in magnetic resonance imaging of the tissue of a living subject which composition comprises:
L-M
wherein M is a metal (II) or (III) ion indepen-dently selected from the group consisting of metals of atomic number 21 to 31, metals of atomic number 39 to 50, the lanthanide metals having an atomic number from 57 to 82, and metals of atomic number 72 to 82, and L is a polydentate organic chelating moiety of structure Ia:

(Ia) wherein Q, J, Y and Z and X and X1 when present are each independently selected from -CH2(C=O)-OH, or -CH2(C=O)NH-CH-(R)-(A);
wherein R in each of Q, J, X, X', Y and Z is inde-pendently selected from hydrogen or an organic structure comprising an alkyl, aryl, substituted aryl, alkylene aryl, alkylene substituted aryl, heteroaromatic, substi-tuted heteroaromatic, alkylene heteroaromatic, or alkyle-ne substituted heteroaromatic group provided that at least one of Q, J, Y and Z is -CH2(C=O)-NHCH(R)-A;
A is independently selected from -(C=O)OR1, -(C=O) -N-(R2)R3, or R4 wherein R4 is independently selected from -CH2-aryl, -CH2-substituted aryl, -CH2CH2-aryl, or -CH2CH2-substituted aryl, provided that when A is -(C=O)OR1 and R1 is hydrogen then R is not hydrogen;
R1, R2 and R3 when present in A in each of Q, J, X, X', Y and Z are independently selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl; and m is selected from 0, 1, 2 or 3, and n is selected from 0 or 1, or the pharmaceutically acceptable salt(s) thereof.

28. The composition of Claim 27 wherein Q and Y are each -CH2(C=O)NHCH(R)COOR1, J, and Z are each -CH2(C=O)OH, and m and n are each 0.

29. The composition of Claim 28 wherein R is -CH2-phenyl, and R1 is hydrogen.

30. The composition of Claim 30 wherein M is a (III) metal ion, Q and Y are each -CH2-(C=O)NHCH(R)-COOH wherein R is selected from benzyl, p-hydroxyphenylmethyl, 2-methyl-naphthyl, or 3-methylindolyl.
J and Z are each -CH2(C=O)-OH, m is 0 or 1, and X is - CH2(C=O)OH and n is 0.

31. The contrast agent of Claim 27 wherein the metal ion is selected from iron (III), cobalt (III), chromium (III), gadolinium (III) or manganese (II) or (III).

32. The composition of Claim 27 useful as an in-jectable contrast agent of a concentration of between about 0.5 and 5000 micromol/kilogram of body weight of a human being.

33. A polydentate organic chelating compound of structure Ia:

(Ia) wherein Q, J, Y and Z and X and X1 when present are each independently selected from -CH2(C=O)-OH, or -CH2(C=O)NHCH-(R)-(A);
wherein R in each of Q, J, X, X', Y and Z is inde-pendently selected from hydrogen or an organic structure comprising an alkyl, aryl, substituted aryl, alkylene aryl, alkylene substituted aryl, heteroaromatic, substi-PCT/US92/0666?

tuted heteroaromatic, alkylene heteroaromatic, or alkyle-ne substituted heteroaromatic group provided that at least one of Q, J, Y and Z is -CH2(C=O)-NHCH(R)-A; and A is independently selected from -(C=O)OR1, -(C=O) -N-(R2)R3, or R4 wherein R4 is independently selected from -CH2-aryl, -CH2-substituted aryl, -CH2CH2-aryl, or -CH2CH2-substituted aryl, provided that when A is -(C-O)OR1 and R1 is hydrogen then R is not hydrogen;
R1, R2 and R3 when present in A in each of Q, J, X, X', Y and Z are independently selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl; and m is selected from 0, 1, 2 or 3, and n is selected from 0 or 1, or the pharmaceutically acceptable salt(s) thereof.

34. The contrast agent of Claim 33 wherein the .alpha.-amino acid(s) when present are selected from the D or L
configuration or mixtures thereof.

35. The contrast agent of Claim 33 with the priviso that at least one R or A comprises an aryl, substituted aryl, alkylenearyl, alkylene substituted aryl, heteroaromatic, substituted heteroaromatic, alkylene heteroaromatic, or alkylene substituted heteroaromatic group.

36. A method of examining human tissue in a diag-nostic manner, which method comprises:
(a) injecting a human being with a contrast agent of Claim 1 in a dose amount having a concentration of the compound of structure Ia of between about 0.5 and 5000 micromol/kg of body weight in the human being;
(b) placing the human being in a magnetic field and irradiating the torso and abdomen of the subject of step (a) with radio-frequency energy such that nuclear magnet-ic resonance can be detected; and (c) analyzing the imaging nuclear magnetic reso-nance signals obtained.

37. A method of preparing a chelate compound of Claim 33 of structure (Ia) which method comprises:
(a) contacting a structure of the formula II:

(II) wherein A and D are CN2(C=O)-OH, with an amino acid or ester of the structure:
H2N-CH(R)-COOR1 or amide of the structure H2N-CH(R)CONR2R3 or a derivative of the structure NH2CH2-CH2-substituted aryl as defined herein above wherein R is an organic structure comprising an alkyl, aryl, alkylene aryl, a heteroaromatic or alkylene heteroaromatic group, and R2 and R3 are independently selected from hydrogen, alkyl having from 1-7 carbon atoms, phenyl or benzyl, and m is selected from 0, 1, 2 or 3, and n is selected from 0 or 1, in an anhydrous dipolar aprotic solvent at between about 50 and 150° for between about 2 and 10 hr; and (b) removing the solvent and recovering the com-pound of structure Ia.

38. The method of Claim 37 wherein the dipolar aprotic solvent is independently selected from dimethylf-ormamide, diethylformamide, hexamethylphosphoramide, dimethylsulfoxide or mixtures thereof; and the heating temperature is between about 90 and 100°C and the time is between about 4 to 7 hr.

39. The contrast agent of Claim 1 wherein M is selected from iron (II), iron (III) or gadolinium (III), Q and Y are each -CH2(C=O)NHCH(R)-(COOR1) wherein R is benzyl and R1 in Q and Y are each hydrogen, or R1 in Q is H and R1 in Y is ethyl, or R1 in Q and Y are each ethyl, and J, X, X' and Z are each -CH2(C=O)OH,

40. The contrast agent of Claim 1 wherein M is selected from iron (II), iron (III), or gadolinium (III), Q and Y are each -CH2(C=O)NHCH(R)-COOR1, wherein R is phenyl, and R1 in Q and Y are each hydrogen, or R1 in Q is H and R1 in Y is ethyl or R1 in Q and Y are each ethyl and J, X, X' and Z are each -CH2(C=O)OH, and m is 1 and n is 0.

41. The contrast agent of claim 1 where m is 1 and n is 0.

42. The contrast agent of Claim 1 wherein R
independently selected from:

, , , ,or .