AU620841B2 - Macrocyclic chelating agents and chelates thereof - Google Patents

Description

PCT

I 0PJ DATE 19/07/89 APPLN. ID 290147 89 PCT NUMBER PCT/EP88/01166 INTERNATIONAL APPLICAT( A0JP DATE 17/08/89 it 1~ ii Ii

F'

(51) International Patent Classification 4 (11) International Publication Number: WNO 89/ 65802 C07N 247/02, 6K4/0A (43) International P Ii o*n DXe: M29 Ju e 1989 29.06.9) (21) International Application Number: PCT/EP88/0I 1166 (8 1) Designate tZ AI ur n paitt), A I BE (European patent), CR (European patent), E (Euro- (22) International Filing Date: 16 December 1988 (16.12.88) pean patent), FR (European patent), GB (European patent), IT (European patent), J P, KR, LU (European patent), NL (European patent), SE (European pa- (31) Priority Application Number: 23217 A/87 tent), SU, US.

(32) Priority Date: 24 December 1987 (24.12.87) Published (33) Priority Country: IT With international search report.

Before the expiration of the time limit for amnending the claims and to be republished in the event of the receipt (71) Applicant (for all designated States except US)l: BRAC- of amendments.

CO INDUSTRIA CHIMICA S.P.A. [IT/IT]; Via E.

Folli, 50, 1-20134 Milano (IT).

(72) Inventors; and Inventors/Applicants (for US only) :FELD ER, Ernst [IT/ IT]; MUSU, Carlo [IT/IT]; FUMAGALLI, Luciano [IT/IT]; UGGERI, Fulvio [IT/IT]; Via E. Folli, 50, 1- 20134 Milano (IT).

(74) Agent: MINOJA, Fabrizio; Studio Consulenza Brevettuale, Via Rossini, 8, 1-20122 Milano (IT), (54) Title: MACROCYCLIC CHELATING AGENTS AND CHELATES" THEREOF

N

3 3- N N B, (I)

-CR

CO-x -CHi cO-Y

(II)

(III)

(57) Abstract Macrocyclic derivatives of l,4,7,l0-tetraazacyclododecane of general formula wherein A is a group of formula in which R is H or alkyl or optionally substituted benzyl or a H(OCH 2

CH

2 14 Mve(OCH 2

CH

2 1 4 or Et(OCH 2

CH

2 14 group, X is O-R 1 in wvhich R, is H or alkyl, hydroxyalkyl, alkoxyalkyl, alkoxyhydroxyalkyl or a polyoxaalkyl group or X is -NRZR 3 in which R 2 and R 3 are H or alkyl, hydroxyalkyl, alkoxyalkyl or alkoxyhydroxyalkyl, and B 1

B

2 and B 3 have the same meanings as A or are H or a group of formula (111), in which R.4 is H or alkyl, Y is a O-R group, wherein Rs is V4, or alkyl, hydroxyalkyl, alkoxyalkyl, alkoxyhydroxyalkyl or a polyoxaalkyl group, or Y is a -NR 6

R

7 group, wherein Rs and R 7 are H or alkyl, hydroxyalkyl, alkoxyalkyl, or alkoxyhydroxyalkyl, said derivatives optionally being salified, and the, complex salts thereof; are used as pharmaceuticals and/or diagnostic 'agents, WO 89/05802 '1PCT/EP88/01 166 MACROCYCLIC CHELATING AGENTS AND CHELATES THEREOF The present invention relates to novel macrocyclic chelating agents deriving from 1,4,7,10-tetraazacyclododecane of general formula I B 3 N N-B I

NI

wherein 2 A is a group of formula CH

-CH

CO-x in which R is H or a C C5 straight or branched alkyl group, or a benzyl group which can be mono- or poly- substituted on the aromatic ring by halogen, hydroxy, carboxy, carbamoyl, alkoxycarbonyl, sulphamoyl, lower alkyl, lower hydroxyalkyl, amino, acylamino, acyl, hydroxyacyl groups, or a group of formula H(OCH 2CH 2) Me(OCH CH ,or Et(OCH CHi) 2 2)1-4- 2 2)1-4- X is a O-R 1 group in which RIis H or a C 1 -C 5 al~kyl, hydroxyalkyl, alkoxyalkyl, alkoxyhydroxyalkyl group, or -2a polyoxaalkyl group having 1 to 15 oxygen atoms and 3 to carbon atoms, or X is a -NR 2

R

3 group in which R 2 and R 3 which can be the same or different, are hydrogen

C

1

-C

6 alkyl, hydroxyalkyl, alkoxyalkyl or alkoxyhydroxyalkyl groups having up to 5 hydroxy groups and B1, B 2 and B3, which can be the same or different, have the same meaning as A or they are H or a group of formula 4 R4 is H or a C -C straight or branched alkyl group, S4 1 Y is a O-R 5 group in which R is H or a 5 l2 grupi whhichC1 20 alkyl, hydroxyalkyl, alkoxyalkyl, alkoxyhydroxyalkyl group, Ior a polyoxaalkyl group having 1 to 15 oxygen atoms and 3 to 45 carbon atoms, or Y is a -NR 6

R

7 group in which

R

6 and R 7 which can be the same or different, are H or

C

1

-C

6 alkyl, hydroxyalkyl, alkoxyalkyl or alkoxyhydroxyalkyl groups having up to 5 hydroxy groups, said derivatives being, if necessary, salified with suitable organic or inorganic bases; with the proviso that when X is O-R 1 in which R1 is H or C 1

-C

5 alkyl, 2anot hydroxyalkyl, alkoxyalkyl, R is4hydrogen, C 1

-C

5 alkyl or H(OCH 2

CH

2 Me(OCH 2

CH

2 1 _4, Et(OCH 2

CH

2 )1- 4 and the complex salts of the abovesaid chelating agents with suitable metal ions in the acid, basic or neutral form or, if necessary, neutralized with inorganic or organic acid or base ions, and eventually chemically conjugated with macromolecules or incorporated in suitable

*S

2 IWO.89/05802 MM M PCT/EP8/01166 3

U

p j s e I carriers.

The present invention also relates to the preparation of compounds of general formula I and of the complex salts thereof, to their uses and, when indicated, to the pharmaceutical and diagnostic compositions thereof.

The chelating compounds of the present invention and the complex salts thereof can have a wide range of applications. No limiting examples of use of said chelating agents are the recovery, separation, selective extraction of r.ual ions even at very low concentrations, their therapeutical use as detoxifying agents in cases of inadvertent bodily incorporation of metals or radioisotopes, their use as ion carriers, or the other ones apparent to those skilled in the art. In such uses the chelating agents may be used directly or often they have been bonded covalently or non-covalently to macromolecules or insoluble surfaces or have been otherwise incorporated into structures that can carry them to specific sites.

In particular the complex salts of the chelating agents of 20 formula I with the metal ions of the elements with atomic numbers of 20 to 31, 39, 42, 43, 44, 49 or 57 to 83 and, optionally, salified by physiologically biocompatible ions of organic or inorganic acids or organic or inorganic bases or aminoacids, are surprisingly suitable for use as contrast agents in medical diagnosis in nuclear medicine and in X-ray and/or ultrasonic diagnosis.

Said derivatives, for the purpose of optimal diagnostic use, can also be bound or incorporated covalently or non-covalently into biomolecules, .macromolecules or molecular aggregates characterized in that they can selective- I W0 89 ~i 11i

I

/05802 4

'I

pCT/EP88/01166,, ly concentrate in the organ or in the tissue under examination.

The imaging of internal structures of living s:,-jects is becoming more and more relevant in medical diagnosis.

Among the most recent techniques, the use of radioisotopes as internal tracers in the organism should be mentioned.

One of the biggest problems connected with the use of radioisotopes is their selectivity of distribution, while another important aspect is their excretion in an acceptable time.

Another imaging technique concerns with the use of ultrasounds to measure the difference in the reflections at the interfaces between tissues of different density.

The administration of a suitable amount of a dense non-radioactive element or metal ion can give such a difference in reflectivity that can emphasize even small otherwise non detectable lesions.

A third diagnostic technique uses nuclear magnetic resonance to create internal images of the human body. In this field, the development of contrast agents is of particular importance for the following reasons: a) to improve the specificity of the diagnosis, b) to identify at an earlier stage small lesions, c) to more precisely define the extension of a tumoral mass, d) to improve the signal to noise ratio and to shorten the time of acquisition of the images, allowing also better use of the instruments, e) to increase the contrast between those contiguous areas (for instance abdominal or pelvic) where it is partip i. r'l WJO 89/05802 PCT/EP88/01166 5 cularly difficult to obtain well defined images, f) to obtain good informations on blood flow and on tissue perfusion.

As far as regards N.M.R. diagnosis, contrast media containing par-'"agnetic complex salts of lanthanides and transition metals have already been claimed for instance in EP 71564 and in US-Pat. 4,639,365, and in patent applications DE 3401052, EP-A 135125, EP-A 130934, DE 3324236, EP-A 124766, EP-A 165728, WO 87/02893, EP-A 230893.

1 However all the till now developed contrast agents for N.M.R. present some problems as far as regards their capacity of influencing the relaxation time of the atomic nuclei involved, their often insufficient selectivity in bonding the metal ion, their stability, their selectivity for the organ under examination: or their biological tolerability. The tendency of many complexes to exchange the central metal ion with trace-metals essential *to the organism or with ions, for example Ca 2 which in vivo are present in relatively great amounts (see on the subject P.M. May "The present status of chelating agents in Medicine", page 233), further limits their possibilities of use. In fact, in case of insufficient stability of the complex, the organis; may be deprived of trace-metals of vital importance and receive undesired and toxic heavy metals such as Gd, Eu or Dy. Although it is true that the toxicity of the complex is often, but not always, lower than the one of the free paramagnetic ion, it is also true that the complexation usually brings a decrease of the magnetic relaxation efficacy, responsible for some contrasting effects.

WO89/05802 PCT/EP88/01166 -6- Several unsolved problems in connection with 'an optimal contrast agent therefore still remain, chiefly concerning: a strong effect on the relaxation time of the relevant nuclei, a high stability of the complex both in solution and in the organism, an adequate water solubility, a specificity of distribution in the various parts of the organism, a suitable rate of elimination from the involved organ and tissue.

One of the most studied paramagnetic metal ions is Gd( in particular when complexed with the chelating agent diethylenetriamino-pentaacetic acid (DTPA) (Runge et al.

(1983) Am. J. Radiol. V 141, p. 1209 and Weinman et al.

(1984) Am. J. Radiol. V 142, p. 619). Said complex salified with D(-)-methyl-glucamine is considered at the moment one of the most satisfying from the point of view of activity, toxicity and of its use in general.

However, in spite of these positive features, this compound cannot yet be considered fully respondent to the characteristics of an optimal contrast agent for various reasons, among which for instance the fact that Gd-DTPA/N-Methyl-D-glucamine is too quickly removed from the blood stream and from the lesions of the tissues under examination. This reduces the time available for obtaining images significant from diagnostic point of view. Moreover the diffusion of' the contrast agent between the. healthy part and the diseased one is often so fast that the contrast between the two regions can be too weak.

To overcome these difficulties, the problem has been approached in many ways among which the most interesting are: .i I W.0 89/05802 P CT/E P88/011166 -7 a) Other chelating agents have been studied, in particular macrocyclic ones, of which the most effective proved to be 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra- Sacetic acid (DOTA). However its complexes continue to present problems analogous to the ones of DTPA.

b) Gadolinium and its chelating agents have been chemically conjugated to macromolecules such as, for instance, proteins (albumin, etc.), immunoglobulins, or to cellulose or other polymeric matrices. On the one hand this generally improved the relaxivity of Gd, but on the other hand it was necessarily accompanied by a sub-optimal dosage, because of limitations in solubility, toxicity and the substitution density of the macromolecules. Furthermore, when one of the ligand sites of the chelating agents is used to form the chemical bond with the macromolecule, there is also normally a reduction in the stability of the resulting complex.

The chelating agents of formula I have shown an excellent scavenging capacity for metal ions even in very diluted solutions. A significant example of said property is the capacity to capture the Cu ion from its aqueous solutions by 2-LT,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)cyclododeca-ne-l-y 7-3-benzyloxypropionic acid, the method of synthesis of which is reported in examples 2 and 3.

With regards to their use in diagnosis, metal complexes with the chelating agents object of the present invention have proved surprisingly satisfying for instance with respect to the requirements for an N.M.R. contrast agent.

Among the complexes particular importance is to be given to the complexes of Gd 3 which distinguish themselves

I

WO 89/05802 PCT/EP88/01166-.

8 for excellent stability, relaxivity and selectivity for the organ or tissue under examination.

These compounds have a wide field of application, allowing administration by intravasal route (for example intravenous, intraarterial, intracoronaric, intraventricular, etc.), as well as intrathecal, intraperitoneal, intralymphatical, intracavital and intraparenchymal routes. Both the soluble and the poorly soluble compounds are suitable for oral or enteral administration, and therefore of particular usefulness for visualization of the gastrointestinal tract. For parenteral administration they are preferably formulated as a sterile aqueous suspension or solution, whose pH can range for instance from 6.0 to 8.5. Said sterile aqueous suspensions or solutions can be administered in concentrations varying from 0.002 to 1.0 molar.

Said formulations, can also be lyophilized and supplied as such, to be reconstituted at the moment of their use. For gastrointestinal use or for injection in body cavities said agents can be formulated as a suspension or a solution containing additives suitable for instance to increase viscosity.

For oral administration they can be formulated according to preparation methods commonly used in pharmaceutical technology, optionally also as a coated formulation so as to have additional protection against the acid pH of the stomach, preventing in that way the release of the chelated metal ions which takes place in particular at the pH va3ues typical of gastric juices. Other excipients, such as sweetening or flavouring agents, can be added according IkO 89/05802 PCT/EP88/01166 9 to known pharmaceutical formulation techniques.

Suspensions or solutions of complex salts can also be r formulated as aerosols to be used in aerosol-bronchography.

Some of the complex compounds of the invention have a surprising organ specificity, in that they particularly concentrate in the liver, in the spleen or, after intralymphatic, intraparenchymal, intramuscular or subcutaneous application, in the lymphatic vases and in the lymph nodes. The resulting contrast between the organ under examination and adjacent tissues permits improved imaging of said organ by N.M.R.

With regard to their use in diagnosis, metal complexes of the chelating agents object of the present invention can also be used as contrast agents in nuclear medicine and for electron spin resonance or echographic analyses.

In these cases however the metal central ions in the chelated complexes are, respectively, a radioisotope for 51 68 111I 99m 140 168 example Cr, Ga, In, Tc, La, Yb or a non-radioisotope able to alter, owing to the density of its solutions, the speed of the ultrasonic waves transmitted and reflected.

I In the compounds of general formula I, A is preferably a 8-hydroxy-X-propionic, B-methoxy-O(-propionic or B-benzylok 25 xy-O-propionic group, optionally esterified or preferably substituted by an amide residue which can be free, monoor bi-substituted by alkyl, hydroxyalkyl, alkoxyalkyl or alkoxyhydroxyalkyl groups.

R can preferably be H or a straight or branched alkyl group, such as a methyl, ethyl, propyl, isopropyl, butyl, C LI WO 89/05802 PCT/EP88/01 166' isobutyl group or a benzyl or a substituted benzyl group as defined in formula 1.

R can also be an acyl or hydroxyacyl group.

R can also be a polyoxaethylene group of formu'la H(OHCHi CH e(OCH- CH- o Et(.HCE) 2 2 2-4 2 2 2-4 2CH2)2-4_ X can be a hydroxy group or also a-O-R 1 group, wherein R 1 1 is as defined in formula I.

Non-limiting examples of R are the following: methyl, 1 ethyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, 1,3-dihydroxyisopropy., polyoxaalkyl groups.

X can preferably be also an hydroxyalkyl amino group of formula -NR 2R 3 in which R 2and R 3are as defined in, dformula I. Non-limiting examples of said groups are the following ones: amino-, 2-hydroxyethylamino-, 2-hydroxypropylamino-, 2,3-dihydroxypropyl amino-, 1,3-dihydroxyisopropylamino-, hydroxy-2-hydroxymethyl-isopropylamino-, N-methyl-N-2-hydroxcyethylamino-, N-methyl-N-2, 3-dihydroxypropylamino-, Li N-methyl-N-l, 3-dihydroxyisopropylamino-, N-methyl-N-2,3,- 6-pentahydroxyhexylamino-, N-2-hydroxyethyl-N-2, 3dihydroxypropylamino-, N-2-hydroxyethyl-N-l, 3-dihydroxyisopropylamino-, N,N-bis-(2-hydroxyethyl)anino-, N,N-bis- 3-dihydroxypropyl)amino-, N,N-bis-(l, 3-dihydroxyisopropyl)amino groups.

In compounds of general formula I, the B 1 B 2 B 3 groups preferably are an aceticn or an C(-propionic group, eventually esterified or substituted by an amido residue which can be in the free form or mono- or bi-substituteai

L

WO 89/05802 PCT/EP8811166 by alkyl, hydroxyalkyl, alkoxyalkyl or alkoxyhydroxyalkyl groups.

R 4can preferably be hydrogen or straight or branched lower alkyl, preferably methyl.

Non-limiting examples for R 4are the following: hydrogen, methyl, straight or branched propyl, butyl and pentyl groups, as defined in formula I.

Y can preferably be a hydroxy group or a -0-R 5 group, in which R 5has th e above defined meanings of formula I.

Non limiting examples of R 5are the following: methyl, ethyl, isopropyl, 2-hydroxyethyl, 2-hydroxypropyl, 1, 3-dihydroxyisopropyl, polyoxaalkyl groups.

Y can preferably be also a hydroxyalkylamino group of formula -NR 6R 7in which R 6and R 7have the above mentioned meanings of formula 1.

Non-limiting examples of said groups are the following ones: amino, 2-hydroxyethylamino-, 2-hydroxypropylamino-, 2,3dihy droxypropyl amino- 1, 3 -dihydroxyi sopropyl amino-, 1,3dihydroxy-2-methUl-isopropylamino-, 2,3, 4-trihydroxy-l-butylamino-, 1,3, 4-trihydroxy-2-butylamiLno-, 1, 3-dihydroxy-2-hydroxymnethyl-isopropylamino-, N-methyl-N-2-hydroxyethylamino-, N-methyl-N-2, 3-dihydroxypropylanino-, N-methyl-N-l, 3-dihydroxyisopropylamino-, N-methyl-N-2, 3,4,5,6- -pentahydroxyhexylamino-, N-2-hydroxyethyl-N-2, 3-dihydroxypropylamino-, N-2-hydroxyethyl-N-l, 3-dihydroxyisopropylamino-, N,N-bis-(2-hydroxyethyl)amino-, N,N-bis-(2,3-dihydroxypropyl)amino-, N,N-bis-( 1, 3-dihydroxyisopropyl)amino groups.

M.etal ions suited to form complex salts with the chelating WO 89/05802 PCT/EP88/01166 12 agents of general formula I are mainly the di- or trivalent ions of the elements having atomic numbers ranging from 20 to 31, 39, 42, 43, 44, 49, or from 57 to 83 and particularly preferred are Fe 2 Fe Cu Cr3 Gd Eu Dy or Mn Among the metal radioisotopes, particularly preferred are 51 68 111 99m 140 168 Cr, Ga, In, Tc, La, Yb.

Preferred inorganic acid anions comprise ions such as chlorides, bromides, iodides or other ions such as sulfate. Preferred organic acid anions comprise ions of acids which are generally pharmaceutically used to salify basic substances, such as acetate, succinate, citrate, fumarate, maleate.

Prefer-2d inorganic base cations comprise alkali metal ions, such as lithium, potassium and sodium, the latter being particularly preferred.

Preferred organic base cations comprise primary, secondary and tertiary amines, such as ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethylglucamine and N-methylglucamine, the latter being preferred.

Preferred amino acid cations comprise, for example, those of lysine, arginine and ornithine.

Non-limiting examples of the macromolecules suited for conjugation with the chelate complexes of the invention are the following: biomolecules, such as hormones (insulin), prostaglandins, steroidal hormones, amino sugars, peptides, proteins (albumin, human serum albumin), lipids, antibodies such as monoclonal antibodies, polysaccharide chains.

The chelated complexes of the invention can also be SWO 89/05802 PCT/EP88/01166 13

I

incorporated into liposomes, used in form of mono- or multi-lamellar vescicles.

The chelating agents of general formula I and the complex salts thereof are preferably prepared by reacting 1,4,7,10-tetraazacyclododecane prepared according to the method of Atkins et al. (JACS 96, 2268 (1974)), rN N N

-N-

with the desired 0-halo-propionyl derivative III R-O-CH -CH-CO-X 2

III

P

i wherein 2 is halogen and R and X have the above defined meanings, to give the addition product IV R-O-CH -CH-CO-X r IV H N Iv

.N

Aj PCT/EP88/011 66 \ur% QO ncQn UJ- 14 or the corresponding polysubstituted products on the nitrogen atoms of II, depending on the excess of III used.

Compound IV can also be obtained, for example, by protecting diethylenetriamine V with a suitable protecting group P, wherein P can be, for example, a phthaloyl group or another appropriate protective group known in the literature Greene: "Protective groups in organic synthesis"- 1980), NH 21/NH,/%1 NH P N NH P 2 2 V VI by alkylating the resulting compound VI with the proper halo-derivative III C 2 VI III (P N-CH -CH N-CH VII S2 2 2

CO-X

and by finally condensing the resulting product VII, after deprotection and subsequent tosylation, with tosyldiethanolamine.

Compound IV or the polysubstituted analogues thereof can in turn be subjected to condensation with the appropriate %-halo-acetic derivative VIII, or with a suitable precursor thereof (such as an ester or a nitrile), WO 89/05802 PCT/EP88/01166 15 R -CH-CO-Y 4 Z VIII wherein Z is halogen and R and Y have the above defined meanings, to give the desired chelating agents of general formula I.

Finally, chelation of the desired metal ion is obtained preferably by reacting the appropriate derivative of formula I with the stoichiometric amount of metal, in form of a salt or an oxide, possibly in the presence of the base or acid amounts necessary for neutralization.

Condensation of II with III is carried out preferably in water or in a dipolar aprotic organic solvent, such as dimethylformamide (DMF) or dimethylacetamide (DMAC) or in a mixture thereof, at a temperature from 30 to 150 0

C,

preferably from 40 to 100 0

C.

Subsequent condensation of IV with VIII can be effected in an aqueous medium or in an organic solvent, in the presence of an appropriate inorganic or organic base, such as sodium hydroxide, potassium hydroxide, potassium carbonate or tetrabutylammonium hydroxide (TBAOH), at a pH ranging from 8 to 12, preferably from 9 to 11. The temperature can range from 40 to 100 0 C preferably from 50 to 70 0

C.

Finally, preparation of the metal complex salt is preferably carried out in water or in an appropriate water-alcohol mixture, while the temperature can range from 25 to 100 0 C, preferably from 40 to 80 0

C.

The choice of the metal ion and, if necessary, of the r- m- WO 89/05802 PCT/'P88/01166- 16 neutralizing ion is strictly related to the use of the resulting complex.

EXAMPLE 1 2-(1,4,7,10-tetraazacyclododecane-l-yl)-3-benzyloxypropionic acid, trihydrochloride.

A) Sodium 2-chloro-3-benzyloxypropionate g of 2-chloro-3-benzyloxypropionic acid (0.396 mol) were suspended in 550 ml of water and neutralized to pH 7 with 10% sodium hydroxide. After stirring for min, the resulting aqueous solution was washed with ethyl ether and evaporated to dryness under vacuum to give the desired compound.

90.6 g sodium 2-chloro-3-benzyloxypropionate (0.383 mol) were obtained.

Yield: 96.7%; Elemental analysis: calc.: C 50.75 H 4.26 Cl 14.98 found: C 50.68 H 4.33 Cl 14.89 B) 2-(1,4,7,10-tetraazacyclododecane-l-yl)-3-benzyloxypropionic acid, trihydrochloride.

A suspension of 17.2 g of 1,4,7,10-tetraazacyclododecane (0.1 mol) and of 71 g of sodium 2-chloro-3-benzyloxypropionate (0.3 mol) in 70 ml of water was j heated to 50 0 C for 24 h. The resulting solution was diluted to 400 ml with water, dropped into 200 ml of 2N HCl, extracted several times with methylene chloride and then was evaporated to dryness under vacuum.

The crude residue was taken up into 400 ml of water and adsorbed on amberlite IR 120, from which it was WO0 89/05: 30 802 PCT/EP88/01 166 17 luted by means of 5N sodium hydroxide. By concet- :ration of the basic eluate 29 g of a residue were Dbtained, which were dissolved in 400 ml of absolute 2thanol at 60 0 C; the solution was acidified with 200 nI of 6N HCI/EtOH and the resulting precipitate was Stirred at 60 0 C for 1 h. After cooling, the solid was filtered and dried to give the desired compound.

33.5 g of 2-(l,4,7,l0-tetraazacyclododecane-l-yl)-3- Denzyloxypropionic acid, trihydrochloride (0.0729 mol.) aere obtained.

Yield: 72.9% m.p. 221-224 0

C

Titres: (NaOH) :96.9% (AgNO 3: 99.0% Elemental analysis: calc.: C 47.01 ;H 7.23 Cl 23.13 ;N 12.18 found C 47.13 ;H 7.32 Cl 22.92 ;N 12.09; TLC: Support: silica gel plate (Merck Eluent: CHCl AcOH HO0= 5 5: 1 Developer Cl 2+ o-Toluidine Rf 0.35 1H-NMR, 13C-NMR and IR spectra agreed with the indicated structure.

EXAMPLE 2 2- 4, 7, 0-tetraaza-7- (l-carboxy-2-benzyloxy-ethyl )-cyclododecane-l-yi7.-3-benzyloxypropionic acid.

A solution of 12 g of 1,4,7,l0-tetraazacyclododecane (0.069 mol) and 82.32 g of sodium 2-chloro-3-benzyloxypropionate (0.348 mol), obtained a~ccording to the process described in example 1-A, in 120 ml of DMF was placed WO 89/05802 PCT/EP88/01166 18 in a sealed vessel and heated to 50 0 C for 30 h.

The salt formed was filtered and the solvent was distilled off under reduced pressure. The residue was taken up into i 300 ml of water, the pH was adjusted to 2.5 with hydrochloric acid, then the mixture was extracted with four 50 ml portions of methylene chloride.

The organic phase was evaporated to dryness and the residue was dissolved in 200 ml of 0.01N HCI and washed with ethyl ether.

The pH was adjusted to 6 with 10% sodium hydroxide and the aqueous solution was evaporated to dryness. The crude residue was taken up into 30 ml of water and adsorbed on amberlite IR 120, from which it was eluted with ammonium hydroxide.

By concentration of the basic eluate, a residue of 7 g was obtained, which was crystallized from water.

5.85 g of 2-(,4,7,10-tetraaza-7(l-carboxy-2-benzyloxyethyl)-cyclododecane-l-yl7-3-benzyloxypropionic acid (0.011 mol) were obtained.

Yield: 16% 170 0 -175°C Elemental analysis: calc: C 63.61 H 7.63 N 10.60 found: C 63.48 H 7.82 N 10.51 Analogously, the following compounds were obtained: 2-L/,4,7,10-tetraaza-4-(l-carboxy-2-benzyloxy-ethyl)-cy clododecane-l-yl -3-benzyloxypropionic acid; S- 2-C,4,7,10-tetraaza-4,7-di(l-carboxy-2-benzyloxy-ethyl)cyclododecane-l-yl7-3-benzyloxypropionic acid.

EXAMPLE 3 2 -jl,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododeca- Wo: i 'i I 89/05802 PCT/EP88/01166 19 ne-l-yl7-3-benzyloxypropionic acid. (Method A).

To a suspension of 23 g of 2-(1,4,7,10-tetraazacyclododecane-l-yl)-3-benzyloxypropionic acid trihydrochloride (0.05 mol), obtained according to the process described in example 1, and 27.8 g of bromoacetic acid (0.2 mol in 100 ml of water about 60 ml of 6N sodium hydroxide were added, under stirring, to reach pH=10. The mixture was heated to 50 0 C for 17 h and the pH was kept at 10 by further additions of 6N sodium hydroxide.

The solution was cooled and applied to amberlite IR 120, from which the product was eluted with 5N ammonium hydroxide. The basic eluate was evaporated to dryness, the resulting crude compound was dissolved in water and the solution was acidified to pH 3 with 5N ECl.

The precipitate was filtered and crystallized from water to give the desired compound.

15.3 g of 2-l,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-yl7-3-benzyloxypropionic acid (0.029 mol) were obtained.

Yield: 58.4% 173 0 C with dec.

Titre: (NaOH) 99.6% (ZnSO4): 99.5% (HPLC) 99.0% Elemental analysis: calc: C 54.95 H 6.92 N 10.68 found: C 54.77 H 6.96 N 10,77 H-NMR, 13C-NMR and IR spectra agreed with the indicated structure.

WO 8905802PCT/EP88/01 166, EXAMPLE 4 2 -Z1, 4, 7, 1 0-tetraaz a-4, 7, 10O-tri (carboxymethyl -cyclododec ane-l-y.7-3-benzyloxypropionic acid (Method B).

A mixture of 10 g of 2-(l,4,7,l0-tetraazacyclododecale-1yl)-3-benzyloxypropionic acid (0.028 mol), obtained according to the process described in example 1 but without formation of the hydrochloride, and 45.57 g of bromoacetic acid (0.112 mol) in 60 ml of water was treated according to the same process as in example 3, to give the desired compound.

7.93 g of 2-Z7,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)cyclIododec ane -y7- 3 -benzyl oxyprop ionic acid (0.015 mol) were obtained.

Yield: 54% 169-1720C with dec.

Titzre: (NaOH) :99.3% (ZnSO 99.5% Elemental analysis: caic.: C 54.95 ;H 6.92 ;N 10.68 found: C 54.71 ;H 7.00 ;N 10.64 The other cheuico-physical characteristics agreed with the ones of the compound obtained according to method A (example 3).

Analogously, the following compounds were obtained: 2-LT,4,7,l0-tetraaza-4,7,10-tri(l-carboxy-ethyl)-cyclododecane-l-y.17-3-benzyloxypropionic acid.

2-ZL,4,7,10-tetraaza-4-(1-carboxy-2-benzyloxy-.ethyl)-7,1o -di (carboxyv',:thyl )-cyclododecane-1-yi?.-3-benzyloxypropionic acid.

2-LT,4,7,10-tetraaza-7-(1-carboxy-2-benzyloxy-ethyl)-7,10 WO 89/05802 PCT/EP88/01 166 -21 -di (carboxyrnethyl )-cyclododecane-l-yLj-3-benzyloxyprop3-onic acid.

-2-/-I,4,7,l0-tetraaza-4,7-di(l-carboxy-2-benzyloxy-ethyl)- 1 O-carboxyrnethyl-cyclododecane-l-y17- 3-benzyloxypropionic acid.

EXAMPLE D(-)-N-methylglucamine salt of the Gd /2-LI,4,7,lo-tetraaza-4, 7, 10-tri(carboxymethyl )-cyclododecane-l-y17-3-benzyloxyprcopionic acid complex.

To a suspension of 100 g of 2-LI,4,7,1Q--tetraaza-4,7,10tri (carboxymethyl )-cyclododecane-l-y17-3-benzyloxypropionic acid (0.19 mol), obtained according to the process described in example 3, in 150 ml of water 36.6 g of D(-)-N-methylglucamine (0.187 mol) were added. 19.47 g of Gd 20 3(0.095 mol) were added to the solution and the resulting suspension was heated to 50 0 C for 4 hours. The reaction mixture was filtered and the pH was adjusted to by means of a 10% aqueous D(-)-N-methylglucamine solution. The resulting solution was then evaporated and dried to give the desired compound.

159 g of D(-)-N-methylglucamine salt of the Gd 14 l0-tetraaza-4 ,7,10-tri(carboxymethyl )-cyclododecane- 1-yl7-3-benzyloxypropionic acid complex (0.182 mol were obtained.

Yield: 95.8% 1370C Titre: (HPLC) :99.3% Elemental analysis: calc.: C 42.56 ;H 5.76 ;Gd 17.99 ;N 8.01 found: C 42.42 ;H 5.96 ;Gd 17.63 ;N 7.92 WO 89/05802 PCT/EP88/01166~ 22 ZC 0=-30zk 20 1.22 0Z 20 6.0' 20= 57 ~~365- -l.3o 36 l.2 ~546 =6.o 589=.57 (C 51 H Analogously, the following compounds were obtained: Dy (3)/2-/T,4,7,10-tetraaza-4,7,lO-tri(carboxymethyl)-cyclododecane-l-yl7-3-benzyloxypropionic acid salt of D(-)-N-rnethylglucanine, obtained with Dy 2 0 3 La /2-./l-,4,7,l-tetraaza-4,7,lO-tri(carboxymethyl)-cyclododecane-l-y17-3-benzyloxypropionic acid salt of D(-)-N-methylglucamine, obtained with La 20 3 Yb (3)/2-ZI,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-y13-3-benzyloxypropionic acid salt of D(-)-N-methylglucamine, obtained with Yb 20 3 EXAMPLE 6 b(-)-N-methylglucamine salt of Gd (3)/2-Z1,4,7,l0-tetraaza-4,7,l0-tri(carboxymethyl)-cyclododecane--y7-3-hydroxypropionic acid complex.

A solution of 92 g of D(-)-N-methylglucamine salt of clododecane-1-y1*7-3-benzyloxypropionic acid complex (0.105 mol), obtained according to the process described in example 5, in 550 ml of water, to which 153 g of palladium on charcoal had been added, was hydrogenated for h at room temperature.

The catalyst was removed by filtration and the aqueous solution was evaporated under vacuum at 50 0 C. Upon drying the residue to constant weight, the desired debenzylated compound was obtained.

67 g of D(-)-N-methylglucamine salt of Gd 3 +)/2-LI,4,7,10-tetraaza-4,7,l0-tri(carboxymethyl)-cyclododecane-1-y17-3- hydroxypropionic acid complex (0.084 WO089/05802 23-PCT/EP88/O1 166 mol) were obtained.

Yield: 80% 1800C (dec.) Elemental analysis: caic: C 36.77 ;H 5.65 ;Gd 20.06 N 8.93 found: C 36.47 ;H 5.47 ;Gd 20.29 N 8.83 2-20 20 6 -70 -~720 ZR2365 1l6.70 503= 11.20 Z-c'(7546 -67 ~589 -58 (C 5% H 02).

Analogously, the following compounds were obtained: -2-(1,4,7,1Q-tetraazacyclododecane-l-yl)-3-hydroxypropionic acid.

2-fl1,4,7,10-tetraaza-4-(l-carboxy-2-hydroxy-ethyl)-cyclododecane-1-y.Lj-3-hydr,-xypropionic acid.

2-/i1,4,7,10-tetraaza-7-(l-carboxy-2-hydroxy-ethyl)-cyclododecane-1-ylJ-3-hydroxypropionic acid.

-2-jI,4,7,10-tetraaza-4,7-di(l-carboxy-2-hydroxy-ethyl)cyclododecane-l-y1L7-3-hydroxypropionic acid.

-2-ZI, 4, 7,10-tetraaza-4- (-carboxy-2 -hydroxy-ethyl) (carboxymethyl )-cyclododecane-l-y17-3-hydroxypropionic acid.

2-lI,4,7,10-tetraaza-7-(l-carboxy-2-hydroxy-ethyl)-4,10di (carboxymethyl )-cyclododecane-1-y7) -3-hydroxypropionic acid.

-2-Fl,4,7,10-tetraaza-4,7-di(1-carboxy-2-hydroxy-ethyl)l0-carboxyrnethyl-cyclododecane-l-yIJ 3-hydroxypropionic acid.

-2-L1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-1-yf/7-3-hydroxypropionic acid.

EXAMPLE 7 2-Cl, 4, 7,10-tetraazacyclododecane-l-yl )-3-benzyloxy-N- 3dihydroxyisopropyl )-propionamide.

PCT/EP88/01166..

ITYO 89/05nO2 24- A) 3-benzyloxy-2--Thloropropionylchloride.

119 g of thionyl chloride (1 mol) were added dropwise to 107.3 g of 3-benzyloxy-2-chloropropionic acid mol) at 30 0 C. After refluxing the reaction mixture for 2 h, 33 g additional thionyl chloride (0.277 mol) were added and the mixture was refluxed for another. mi n.

Excess thionyl chloride was distilled off under reduced pressure and the desired compound was distilled under vacuum.

95.8 g of 3-benzyloxy-2-chloropropionylchloride (0.41 mol) were obtained.

Yield: 82% 125-131WC; 0.05 mbar Titre: (reduction with Zn) 99.9% (Argentometric) 96.0% Elemental analysis: calc: C 51.53% ;H 4.32% Cl 30.42% found: C 51.30% ;H 4.46% 29.481 1H -NMR, 13C-NMR and IR spectr~a agreed with the indicated structure.

B) 2-Chloro-3-benzyloxy-N-(l, 3-dihydroxyisop,-opyl)-propionamide.

A solution of 70 g of 3-benzyloxy-2-chloropropionyl.

chloride (0.3 mol) in 150 ml of tetrahydrofuran was added dropwise during about 2 h to a solution of 32.6 g of 2-amino-1,3-dihydroxyisopropane (0.36 mol) in 150 mil of water and 250 ml of tetrahydrofuran. During the addition of the chloride, the pH of the solut~on was kept constant at 10 by addition of 6N sodium WO 89/05802 PCT/EP88/01166 25 hydroxide.

To the reaction mixture 230 ml of water were added.

Upon concentration to 450 ml a white -product precipitated, which was filtered and crystallized from water, after treatment with Carbopuron 4N to give the desired compound.

62.2 g of 2-chloro-3-benzyloxy-N-(l,3-dihydroxyiSopropyl)-propionamide (0.218 mol) were obtained.

Yield: 72.6% 133-135 0

C

Titre: (Argentometric) :99.8% Elemental analysis: calc: C 54.27 H 6.30 Cl 12.32 N 4.87 found: C 54.19 ;H 6.38 Cl 12.24 N 4.84 ;H 0.22 HPLC: 99% 1H-NMR, 13C-NMR and IR spectra agreed with the indicated structure.

C) 2-(l,4,7,10-tetraazacyclododecane-1-yl)-3-benzyloxy- 3-dihydroxyisopropyl )-propionamide, trihydrochloride.

51.6 g of 1,4,7,20-tetraazacyclododecane (0.3 mo).

and 258.75 g of 2-chloro-3-benzyloxy-N-( 1, 3-dihydroxyisopropyl)-propionanide (01.9 mol). obtained according to the process described in example 7-B, were reacted at 70 0 C in DMF for 24 h.

After evaporation of the solvent under vacuum, the residue was taken up in water and adsorbed on an ion exchange resin IR 120, from which it was eluted by means of SN ammionium hydroxide.

WO 89/05802 PCT/EP88/01166,.

26 The ammonia solution was evaporated to dryness and the residue was transformed into the corresponding trihydrochioride, as described in example 1.

63.84 g of 2-(l,4,7,l0-tetraazacyclododrcane-l-yl)-3benzyloxy-N-( 3-dihydroxyisopropyl )-propionamide, trihydrochioride (0.120 mol) were obtained.

Yield: 40.0% rn.p.: 125 0 C (dec.) Elemental analysis: calc: C 47.33 ;H 7.56 ;Cl 19.96 ;N 13.14 found: C 47.41 ;H 7.68 ;Cl 19.85 ;N 13.08 HPLC: 97.6% Analogously, the following compounds were obtained: -2-(l,4,7,l0-tetraazacyclododecane-1-yl)-3-benzyloxy-pro- 8 pionamide.

-2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-benzyloxy-N-(2hydroxyethyl )prioade -2-(J,4,7,10-tetraazacyclododecane-1-yl)-3-benzyloxy-N- I 3-dihydroxypropyl )-propionamide.

-2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-benzyloxy- N,N-di (2-hydroxyethyl)-propionamide.

EXAMPLE 8 2-Li, 4, 7' 0-tetraaza-4, 7, 10-tn (carboxymethyl )-cyclododecae1y:--bnyoyN(1, 3-dihydroxyisopropyl )-propionamide.

A mixture of 16 g of 2-(1,4,40-tetraazacyclododecanelI -yl )-3-benzyloxy-N- 3-dihydroxyisopropyl )-propionamide (0.038 niol), obtained according to the process described in example 7, and of 21.13 g of bromoacetic acid (0.152 mol) in 100 ml of water was reacted by the same process as described in example 3, to give the desired compound.

.WO 89/058 :02 PCT/EP88/01 166 -27 12.4 g of 2Zl1,4,7,10-tetraaza-4,7,l0-tri(carboxymethyl)cyclododecane-l-y17-3-benzyloxy-N- 3-dihydroxyisopropyl) propionarnide (0.0207 mol) were obtained.

Yield: 54.4% 137 0 C (dec.) Titre: (NaOH) 98.8% Elemental analysis: calc.: C 53.98 ;H 7.72 ;N 11.66 found: C 53.91 ;H 7.85 ;N 11.59 Analogously, the following compounds were obtained: 2-Z1,4,7,l0-tetraaza-4,7,l0-tri(carboxyrnethyl)-cyclododecane-l-y17-3-benzyloxy-propionamide.

2-Z1,4,7,l0-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-ylj7-3--benzyloxy-N-( 2-hydroxyethyl )-propionamide.

2-Z-1,4,7,l0-tetraaza-4,7,1Q-tri(carboxymethyl)-cyclododecane-l-yl7-3-benzyloxy-N- 3-dihydroxypropyl )-propionamide.

2-gI,4,'7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-1-l:--3-benzyloxy-N,N-di( 2-hydroxyethyl)-propionamide.

EXAMPLE 9 clododecane-l-ylJ-3-benzyloxy-N-(l, 3-dihydroxyisopropyl propionanide.

To a suspension of 8 g of 2-Ll1,4,7,l0-tetraaza--4,7,l0-tri- (carboxymethyl )-cyclododecane-1-yl7-3-benzyloxy-N- 3-dihydroxyisopropyl)-propionamide (0.013 mol), obtained according to the process described in ex:ample 8, in 30 ml of water 1.33 g of Gd 2 0 3 (0.0065 mol) were added and the mixture was reacted at 50 0 C according to the procedure of WO 89/05802 PCT/EP88/01166'- -28 example The resulting solution was evaporated to dryness to give the desired product.

49 g of Gd /2-Zl,4,7,1o-tetraaza-4,7,10-tri(carboxymethyl )-cycjododecane-l-y1.7-3-berizyloxy-N-(l, 3-dihydroxyisopropyl)-propionamide (0.012 mol) were obtained.

Yield: 92.3% Elemental analysis: caic.: C 42.95 ;H 5.74 ;N 9.28 found: C 42.87 ;H 5.80 ;N 9.23 HPLC: 97.5% EXAMPLE Gd /2-Z'I,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-y17-3-hydroxy-N-(1, 3-dihydroxyisopropyl )-propionamide.

1(3+) 9 g of Gd /2-/l,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-ylj-3-benzyloxy-N-(1, 3-dihydroxyisopropyl)-propionamide (0.012 mol), obtained according to the process described in example 9, were dissolved in ml of water. After addition of 15 g of 5% palladium on charcoal, the solution was hydrogenated according to the procedure of example 6 to give the desired compound.

6.22 g of Gd (3)/2-Z1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-y17/-3-hydroxy-N-(1, 3-dihydroxyisopropyl)-propionamide (0.0096 rnol) were obtained.

Yield: Elemental analysis: caic: C 36.13 ;H 5.61 ;N 10.53 found: C 36.06 ;H 5.64 ;N 10.48 Analogously, the following compounds were obtained: 7 WO 89/05802 PCT!IEP88/0 1166 29 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-hydroxy-propionamide 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-hydroxy-N-(2hydroxyethyl )-propionamide.

2-(J,4,7,10-tetraazacyclododecane-1-yl)-3-hydroxy-N-(2,3dihydroxypropyl )-propionamide.

2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-hydroxy-N,N-di- (2-hydroxyrnethyl )-propionanide.

-2-r/I,4,r7, 10-tetraaza-4, 7, 1Q-tri (carboxymethyl)-cyclododecane-1-y17-3-hydroxy-propionamide.

2-J, 4, 7,10-tetraaza-4, 7, 10-tri (carboxymethyl) -cyclododecane-1-y17/-3-hydroxy-N- (2-hydroxyethyl )-propionanide.

7,10-tetraaza-4, 7, 10-tri (carboxymethyl) -cyclododecane-1-y17-3-hydroxy-N-(C2, 3-dihydroxypropyl )-propionanide 2-L,C,4, 7,10-tetraaza-4, 7, 10-tri (carboxymethyl) -cyclododecane-l-yi7--3-hydroxy-N, N-di (2-hydroxyethyl )-propionanide.

EXAMPLE 11 Determination of the relaxivity of the compounds of the present invention.

Operative conditions Apparatus: Observation frequency: Temperature: MINISPEC PC 120 (BRUKER) 20 MHz (proton) 39 0 C, with pre-therrnostatization of the NMR test tube for min at the operative temperature in the range from 0 to 5 mM with the following specific measuring points: 0/0.1/0.2/0.5/1.0/2.0/5.0 mM D) Concentrations t WO 89/05802 PCTEP88/01166 30 E) Solvent: 0.154 M NaOH water F) pH: 7.3, to be checked again potentiometrically before the relaxation measurement.

Longitudinal relaxivity (R 1 measurements were calculated using the "Inversion Recovery" sequence with an 8 point minimum and a 3 parameter fit, according to the program provided for the MINISPEC 120 BRUKER instrument, by which measurements were taken.

Transverse relaxivity (R 2 measurements were calculated using the sequence of Carr, Purcell, Meiboom and Gill, according to the program provided for the MINISPEC 120 BRUKER instrument, by means of which measurements were taken, adjusting the apparatus in such a way as to observe the decay of the signal to about 1/3 of the starting value, with a score number higher or equal to 10 and a 2 parameter fit.

In table I, R and R 2values calculated for compounds A and B in comparison with Gd/DTPA neutralized with N-methylglucamine, are reported as non-limiting examples.

PCT/EP88/01166 WO 89/(

CUM

31 TABLE I A BGd/DTPA (mM.s) 1 (mM.S) 1 R 1 4.15+0.01 3.72+0.01 4.08+0.01 R 2 5.67+0.02 5.06+0.01 5.15+0.02 A Gd (3)/2-/-1,4,7,10-tetraaza-4,7,10-tri(carboxynethyl)-cyclododecane-l-y1]-3-benzyloxypropionic acid, neutralized with N-methylglucamine.

B =Gd (3)/2-Zl1,4,7,10-tetraaza-4,7,l0-tri(carboxymethyl )-cyclododecane-l-yl]-3-hydroxypropionic acid, neutralized with N-methylglucamine.

neutralized with N-methylglucamine; R 1and R 2values were determined in aqueous solvent.

EXAMPLE 12 Preparation of liposomes incorporating the Gd (3)/2-LI,4,7,10-tetraaza-4,7,l0--tri(carboxymethyl)-cyclododecane-l-y17-3-benzyloxypropionic acid complex, neutralized with N-methylglucamine.

An anhydrous lipidic mixture was prepared, having the following composition: egg phosphatidyicholine 75 mol and cholesterol 25 mol using the REV method Szoka et al., (1978), Proc. Natl. Acad. Sci. U.S.A. 75,4194).

400 mg of said mixture were dissolved in 35 ml of chloro- WO 89/05802 PCT/EP88/01166 32 form to which 10 ml of a 0.05 M solution of N-methyl- D-glucamine salt of Gd (3+)/2-,4,7,10-tetraaza-4,7,10tri(carboxymethyl)-cyclododecane-l-yl7-3-benzyloxypropionip complex acid were added dropwise under sonication.

When the addition was over, sonication was continued for min, Then the crude compound was heated to 50 0 C and the solvent was evaporated under vacuum. The resulting gelly residue was suspended in a 1% NaCl solution and freed from unincorporated chelate by means of five consecutive centrifugations and resuspension steps (26.000 g/10 min).

EXAMPLE 13 Determination of LD 50 in the mouse by intravenous administration of the compounds of the present invention.

In table II are reported, as non-limiting examples, the LD50 values for compounds A and B of the present invention, in comparison with GdC13 and with Gd/DTPA neutralized with N-methylglucamine.

TABLE II in the mouse* in mmol/kg intravenous GdC1 3 0.28 (0.24 0.32) Gd/DTPA** 4.8 (4.47 5.16) A 8.8 (7.79 9.94) B 13.1 (12.2 14.1) WO 89/05802 PCT/EP88/01166 33 male and femal.e mice were used, Strain: Cr1 :CDl(I.CR)BR **=N-methylglucamine salt.

A Gd /2-Z1i,4,7,l0-tetraaza-4,7,10-tri(carboxymethyl )-cyclododecane-l-y17-3-benzyloxypropionic ai, neutralized with N-methylglucamine.

B Gd (3)/2-Cl,4,7,l0-tetraaza-4,7,lO-tri(carboxymethyl)-cyclododecane-l-y17-3-hydroxypropionic acid, neutralized with N-methylglucamine.

Table II shows that, in this pharmacological test, gadolinium complexes with the macrocyclic chelating agents of the invention have substantially decreased toxicities with respect to both GdCl 3and Gd/DTPA.

EXAMPLE 14 Preparation of a solution of D(-)-N-i~ethylglucamine salt of Gd 2-Z 1,4,7,lO-tetraaza-4,7,l0-tri(carboxymethyl)- -cyclododecane-1-y17-3-benzyloxypropionic acid complex.

436.8 g (0.500 mol) of the compound obtained according to the procedure described in example 5 were dissolved in 300 ml of pro iniectione water. The solution volume was taken to 500 ml by addition of water then the solution was filtered, put in vials and rerilized.

EXAMPLE Preparation of a solution of D(-)-N-methylglucamine salt of Gd 2-Zl,4,7,10-tetraaza-4,7,l0-tri(carboxymethyl)- -cyclododecane-1-ylJ-3-hydroxypropionic acid complex.

398.8 g (0.500 mol) of the compound obtained according to the procedure described ILn example 6, were dissolved in 300 ml of water pAi.. The solution volume was taken to 500 ml by addition of water then the solution was WO 89/05802 P CT/E P83/0 1 166 34

I

filtered, put in vials and sterilized.

EXAMPLE 16 Preparation of a solution of D(-)-N-methylglucamine salt of Gd(3+)/2-/I,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)cyclododecane -l-yl7-3-benzyloxypropionic acid complex.

218.4 g (0.250 mol) of the salt cited in example 14 were dissolved in 260 ml of water 0.6 g of ascorbic acid were added and the solution was diluted to 500 ml with water p.i. The solution was sterilized by filtration and put in vials.

EXAMPLE ],7 Preparation of a solution of the D(-)-N-methylglucamine salt of Gd 2-L/,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-yl7-3-benzyloxypropionic acid complex.

218.4 g (0.250 mol) of the salt cited in example 14 were dissolved in 200 ml of water 0.45 g LL tromethamine hydrochloride were added and the solution was diluted to 500 ml with water p.i. The solution was filtered, put in vials and sterilized.

EXAMPLE 18 Preparation of a solution of the D(-)-N-methylglucamine salt of 2-LT,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-yl7-3-hydroxypropionic acid complex.

199.4 g (0.250 mol) of the salt cited in example 15 were dissolved in 200 ml of water 0.6 g of ascorbic acid were added and the solution was diluted to 500 ml with water p.i. The solution was sterilized by filtration and put into vials.

1, 7 ,WO 89/05802 PCT/EP88/01166 35 EXAMPLE 19 Preparation of a solution of the D(-)-N-methylglucamine salt of Gd( 2-/l,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclododecane-l-yl7-3-hydroxypropionic acid complex.

199.4 g (0.250 mol) of the salt cited in example 15 were dissolved in 200 ml of water 0.45 g of tromethamine hydrochloride were added and the solution was diluted to 500 ml with water p.i. The solution was filtered, put into vials and sterilized.

w_

Claims (5)

1. 1,4,7,l0-tetraazacyclododecane of general formula I wherein A is a group of formula CH 2 -0-R -CH Co-.x in which R is H or a C 1-C 5straight or branched alkyl group:. or a benzyl group which can be mono- or poly-substituted on the aromatic ring by halogen, hydroxy, carboxy, carbamoyl, alkoxycarbonyl, suiphamoyl, lower alkyl, lower hydroxyalkyl, amino, acylamino, acyl, hydroxyacyl groups, or a group of formula H(OCH 2CHR 1- Me(OCH 2CHR2)1-4, or Et(OCH 2CH X is aO-R 1group in which R 1is Hor aC hydroxyalkyl, alkoxyalkyl, alkoxyhydroxyalkyl group, or a polyoxaalkyl group having 1 to 15 oxygen atoms and 3 4t. ecy c

37- to 45 carbon atoms, or X is a -NR 2 R 3 group in which R 2 and R 3 which can be same or different, are hydrogen, C 1 -C 6 alkyl, hydroxyalkyl, alkoxyalkyl or alkoxyhydroxyalkyl groups having up to 5 hydroxy groups and B 1 B 2 and B 3 which can be the same or different, have the same meaning as A or they are H or a group of formula 4 co- SCO-Y II I. in which R 4 is H or a CI-C 5 straight or branched alkyl group, I Y is a O-R 5 group in which R. is H or a C-C S 20 alkyl, hydroxyalkyl, alkoxyalkyl, alkoxyhydroxyalkyl group, or a polyoxaalkyl group having 1 to 15 oxygen atoms and 3 H to 45 carbon atoms, or Y is a -NR 6 R 7 group in which R 6 and R 7 which can be the same or different, are H or C 1 -C 6 alkyl, hydroxyalkyl, alkoxyalkyl or alkoxyhydrorvalkyl groups having up to 5 hydroxy groups, said derivatives being, if necessary, salified with suitable organic or inorganic bases, as well as the chelates of compound of formula I with i suitable metal ions, which chelates, if necessary, can in Ti -37a turn be neutralized with inorganic or organic acid or bases; with the proviso that when X is O-R 1 in which R 1 is H or C 1 -C 5 alkyl, hydroxyalkyl, alkoxyalkyl, R is nco;t hydrogen, C 1 -C 5 alkyl or H(OCH 2 CH 2 1 4 Me(OCH 2 CH 2 1 4 Et(OCH 2 CH 2 1 4 2. 1,4,7,1O-Tetraazacyclododecane derivatives of general formula II V. SR*9 WO 89/05802 PCr/EP88101166 38 2-o-cS 2 -CS-co-x CHNN-CE I Y-OC CO -Y -;A-0O-Y wherein R,R 4 and Y have the meanings defined in claim 1, and the chelates thereof with appropriate bi- or trivalent ions of metal elements having atomic numbers from 20 to 31, 39, 42, 43, 44, 49 or from 57 to 83. 3. l,4,7,10-Tetraazacyclododecane derivatives of general formula IIT R-O-CH 2 -CH-CO-X N HOOCCH2- N-H 2 -CO0H I NJ ~co wherein R and X have the meanings defined in claim 1, -and the chelates thereof with appropriate bi- or trivalent ions of metal elements having atomic numbers from 20 to 31, 39, 42, 43, 44, 49 or from 57 to 83. 4. Serine derivatives of general formula IV WVO 89/05802 PCT/EP88/O1 166 39 Ra -o-Ca 2 -CH-CO-x I 2 -N N-CH 2 -COH IV N wherein R 8is H or benzyl, X i is OH, -NH 2 NHCH 2CH 2OH, -NHCH(CH 2 OH) 2 -NHCH 2CH(OHC H -N(CH 2CH 2OH)2 -NH-CH 2-CH(OH)-CH 2OCH or -NH-CR 2-CR(OR)-CH(O)-CH 2 OH, and the chrelates thereof with appropriate bi- or trivalent ions of metal elements having atomic numbers from 20 to 31, 38, 42, 43, 44, 49, or from 57 to 83. 5. A chelate of a compound as claimed in claims 1-4, in which the chelate metal ion is Fe (3 Cu2+ Gd EU Dy or Mn 6. Chelates as claimed in claimu 2 with the ions of the following radioisotopes: 51Cr, 68Ga, ilIn, 9MTc, 18Yb. 7. A compound as claimed in claims 1-6, selected from the group consisting of: 2-(l,4,7,10-tetraazacyclododecane-1-yl)-3-benzyloxypro- picnic acid, 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-hydroxypropio- nic acid, 2-/Ti,4,7,l0-tetraaza-4-(l-carboxy-2-benzyloxy-ethyl)- cyclododecane-1-yi7-3-benzyloxypropionic acid, 2-LI, 4,7, l0-tetraaza-4-( l-carboxy-2-hydroxy-ethyl)- cyclododecane-1-ylJ-3-hydroxypropionic acid, OL WO 89 A /05802 PCT/EP88/01166 40 2-Z/1,4,7,10-tetraaza-7-(1-carboxy-2-befzl~oxy-ethyl)- cyclododecane-1-y17-3-benzyloxpropiolic acid, -2-/I1,4,7,10-tetraaza-7-(l-carboxy-2-hydroxy-ethyl)- cyclododecane-1-yIJ--3-hydroxypropionic acid, 2-L1,4,7,1Q-tetraaza-4,7-di(l-carboxy-2-belzyloxy-ethyl)- cyclodcviecane-1-y27/-3-benzyloxypropionic acid, 2-Zl,4,7,10-tetraaza-4,7-di(l-carboxy-2-hydroxy-ethyl)- cyclododecane-1-y17/-3-hydroxypropionic acid, 2-/1,4,7,10-tetraaza-4-(l-carboxy-2-benzyloxy-ethyl)- 7, 10-di(carboxyrnethyl)-cyclododecane-1-y17-3-beizyloxy- propionic acid, 2-,I,4,7,10-tetraaza-4-(l-carboxy-2-hydroxy-ethyl)-7,10- di (carboxymethyl )-cyclododecane-1-ylj-3-hydroxypropio- nic acid, 2-Cl,4,7,1Q-tetraaza-7-(l-carboxy-2-benzyloxy-ethyl)- 4, 10-di (carboxyrnethyl )-cyclododecane-1-y17-3-berizyloxy- propionic acid, 2-L,4,7,10-tetraaza-7-(1-carboxy-2-hydroxy-ethyl)- 4, 10-di (carboxymethyl) -cyclododecane-1-y17-3-hydroxypro- pionic acid, 2-LT1,4,7,10-tetraaza-4,7-di(l-carboxy-2-benzyloxy-ethyl)- lO-carboxymethyl -cyclododecane-1-yl7-3 -benzyloxypropionic acid, 2-L_1,4,7,10-tetraaza-4,7-di(l-carboxy-2-hydroxy-ethyl)- 10-carboxymethyl-cyclododecane-1-yLj-3-hydroxypropionic acid, 2-LT,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclodo- decane-1-yj17-3-benzyloxypropionic acid, 2-ZlI,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclodo- decane-1-yr7-3-hydroxypropionic acid, ~WO89O58O2PCT/EP88/O1 166 IWO 8/0580

41- 2-(1,4,7,10-tetraazacyclododecafle-1-yl)-3-belzyloxy-pro- pioriaride, 2-(1,4,7,1O-tetraazacyclododecane-1y)-3hydroxy-pro- pionarnide, 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-beflzyloxy-N- A (2-hydroxyethyl )-propionamide, 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-hydroxy-N-(2- hydroxyethyl.) -prop jonarnide, 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-benzyloxy-N- (1,3-dihydroxyisopropyl)-propionamide, 2-(1,4,7,10-tetraazacyclododecane-1-yl)--3-hydroxy-N- 3-dihydroxyisopropyl )-propionamide, V- 2-(1,4,7,10-tetraazacyclododecane-1-yl),-3-benzyloxy-N- 3-dihydroxypropyl )-propionanide, 2-(1,4,7,10-tetraazacyclododecane-1--yl)-3-hydroxy-N- 3-dihydroxypropyl )-propionamide, 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-benzyloxy- N,N-di( 2-hydroxyethyl)-propionanide, 2-(1,4,7,10-tetraazacyclododecane-1-yl)-3-hydroxy-N,N-di- (2-hydroxyethyl)-propionamide, 2-ZT,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclodo- decane-1-y.17-3-benzyloxy-propionanide, 2-L,4,7,10-tetraaza-4,7,10-tri(carboxyrethyl)-cyclodo- decane-1-yr7-3-hydroxy-propionamide, 2-LI,4,7,1O-tetraaza-4,7,10-tri(carboxymethyl)-cyclodo- decane-1-Y.17-3-benzyloxy-N- (2-hydroxyethyl )-propionamide, 2-LT, 4,7, 10-tetraaza-4, 7, decane-1-y.17-3-hydroxy-N- (2-hydroxyethyl) -propionamide, 2-/ 1 ,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-cyclodo- decane-1-yi'7-3-benzyloxy-N-(1, 3-dihydroxyisopropyl)-pro- II

42- 1 pionamide, 2-[l,4,7,l0-tetraaza-4,7,10-tri(carboxyrethyl)- cyclododecane-1-yl]-3-hydroxy-N-(1,3-dihydroxyisopropyl)- propionamide, 2-[1,4,7,0-tetraaza-4,7,10-tri(carboxymethyl)- cyclododecane-1-yll-3-benzyloxy-N-(2,3-dihydroxypropyl)- propioramide, 2-[1,4,7,10-tetraaza-4,7,10-tri(carboxyrethyl)- cyclododecane--yl]-3-hydroxy-N-(2,3-dihydroxypropyl)- propionamide, 2-[l,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)- cyclododecane-1-yl]-3-benzyloxy-N,N-di(2-hydroxyethyl)- propionamide, 2-[1,4,7,10-tetraaza-4,7,1O-tri(carboxymethyl)- cyclododecane-l-ylI-3-hydroxy-N,N-di(2-hydroxyethyl)- propionamide, the respective chelate complexes with Fe( 3 Cu( 2 j (3C) 3t Mn Gd Dy Yb and the corresponding salts with D(-)-N-methylglucamine. 8. A method for the preparation of metal chelates of compounds of formula I of claim 1, which consists in reacting a salt or a metal oxide selected from the ones having atomic numbers from 20 to 31,39,42,43,44,49 or from 57 to 83 with a compound of formula I of claim 1, possibly in the presence of the acid or base amount necessary for neutralization. 9. A diagnostic preparation containing a physiologically acceptable complex salt in which the chelating agent is a compound of general formula I according to claim I and the 'SeEiR, 14 I IE a"M

43- metal ion is selected from the group of the elements having atomic numbers comprised from 20 to 31, 38, from 42 to 44, 49 or from 57 to 83, together with if necessary, an ion of an inorganic or organic acid or base, a suitable physiologically acceptable carrier, and eventually, physiologically acceptable additives. A diagnostic preparation according to claim 9, in which the complex salt is present in a concentration from 0,002 to 1 mol/1. 10 11. An N.M.R diagnostic method, wherein a diagnostic composition according to claim 9, in which the complex salt contains as the metal ion one of those according to claim S' 5, is administered to a patient. 12. A diagnostic method which can be used in nuclear 15 medicine, wherein a diagnostic composition according to .claim 9, in which the complex salt contains as the metal ion-a radioisotope according to claim 6, is administered to a patient. S S DATED SEPTEMBER 20 1991 BRACCO INDUSTRIA CHIMICA S.p.A. By their Patent Attorneys KELVIN LORD AND COMPANY PERTH, WESTERN AUSTRALIA INTERNATIONAL SEARCH REPORT tnternstlonal Application No PCT/EP 88/01166 1. CLASSIFICATION OF SUB3JECT MATTER (it several classification symbol% apply, Indicate till) According to International Patent Classification (IPC) or to both National Classiication and IPC PC 4 IC 07 D 257/02; A 61 K 49/00; G 01 N 24/02 11. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbols 'PC 4 A 61 K 49/00; C 071D 257/00 Documentation Searched other than Minimum Documentation to the Eatent that such Documents ate Included In the Fields Searched I III. DOCUMENTS CONSOE RO TO NE RELEVANT$ Category 1 j citation ol Document. is with Indication, where appropriate, of the relevant pssages 13 Relevant to claim No. A,P EP, A, 0255471 (SCHERING AG) 3 Febriiary 1988, 1-3,5,8,9 see claims; examples 1-7,22 A WO, A, 87/06229 (GUERBET 22 October 1-3,5,9,10 1987, see claims; page 1 A DE, A, 3401052 (SCHERING AG) 26 July 1984, 1-3,5,9,10 see claims 1,20,24,36 (cited in the application) A EP, A, 0232751 SQUIBB SONS, INC.) 1-3,5,9 19 August 1987, see claims 1,16,17; page 2, line 52 page 3, line 14 A S, A, 4639365 SHERRY) 27 January 1 3,5,9 1987, see abstract I(cited in the application) A EP, A, 0124766 (SCHERING AG) 14 November 1984,1-3,5,9 see page 7, last two lines -page 9 (ctdin the application) *Special categories of cited documentst Is IT later document publishied after the International filing date document defining the general state of the art which to not or priority date and not In Conflict with the oaplication but conideed o b ofparicuar eleant cited to understand the principle or theory underig the C:Aodora tobe o paticuar elevnceinvention *eflier document but pubised on or ato the Intetneationsk document at particular relevance' the claimed Invention filing data cannot be coneidered novel Of Cannot be considered to OC d omnt whiCh may throw doubts on priority cloimle) or Involve an iventive clop which Is cited to establish the publication date at another 'Y document of partlicular lrelovsnco* the claimed Invention citation or other special reason (as specified) Cannot be Considered to Involve an Inventive atop when the document referrIng to an oral disclosure, use, exhibition or document is Combined with one or mote Other such docu- other means mania, such Comaination being obvious to a person skilled document published prior to the International filing date but In the art. later than the Priority date Claimed &'document member of the same petent family IV._CERMTIFICATION_____________ Dae of Who Actual Completion of the international search jDate of Malting of this International Search Report 17th March 1989 1- 3. 05. R ET1111 i JROP earcin A t Nt OFFICE-f uti Me ForM PCTIISA12tO second shoot) (January 1"3) Intarnationi~ Application No. PCT/EP 88/01166 -2- [FURTHER INFORMATION CONTINUED itROM THE SECOND SHEET APItrainlJournal of Radiation/ 1-3,5 W" I.i Part B, vol. 15, no. 1, 1988 Marsh Barton, Exeter (GB) J.F. Desreux et al.: "Highly stable lanthanide macrocyclic complexes: in search of new contrast agents for NMR imaging", pages 9-15 Angewandte Chemie, vol. 88, no. 22, 1976, H. Stetter et al.: IlKomplexbildung mit Tetraazacycloalkan-N,N' ,N",N'-tetra- essigsd.uren in Abh~.ngigkeit von der Ring- gr~sse"l, page 760 Aj 1-3 V.rXO3SERVATIONS WHERE CVRTAIN CLAIMS WERE FOUND UNSEARCHABLE'I This International search report has not been established In respect of certain claims under Article 17(2) lor the following reasons: i0Claim numnbers because they relate to subject matter not required to be asrched by this Authority, namely: xx 11,12 Please see Rule 39.1 (IV) PCT: methods for treatment of the human o~r animal body by surgery or therapy, as well as diagnostic methods. 24o Claim numiber because they relate to Pinrs of the International application that do njot comply with the Prescribed require. menia to such an extent that no meaningtul International search can be carried out, specitiauy: Claim niumbrs because trey awe dependent claims and are not drafted in acordanice with the second aN third seriteol of PCT Rule 6.4(m). V1.Q OUSER31FVATIONS WHERE UNITY OF INVENTION 12 LACKING: This International Searching Authority found multiple Invention* In this International application as fllows- AsI all required additional search lees were timely paid by the applicant, this International search report covers all searchable claims of the International application. As only some of the required additional search lees were timely paid by the applicant, this International search. report covers only those claims of the International application for which fees were Paid, specifically claims- No required additional search fees were timely paid by the applicant Consequently, this International search report is restricted to the invention (list mentioned In the claims., it Is covered by claim numbers: 13As 41I seatchableclaims could be searchild without off ort justityltro en additional fee, the International Searching Avthohy did not Inivite payment of any additional fee. Ramon~ an Protest QThe additonal serch (ees were acco'.vpanied by spollcantle praoest QNo protest liternpanlad the payeilt of additional search loee. FemPCTASA2IO (oupolonmentall sheet (Jem-ry 1643) A i 1 t ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. EP 8801166 SA 25969 This annex liss the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 19/04/89 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) d: EP-A- 0255471 03-02-88 DE-A- AU-A- JP-A- 3625417 7621787 63041468 11-02-88 04-02-88 22-02-88 WO-A- 8706229 22-10-87 FR-A,B 2596992 16-10-87 AU-A- 7235087 09-11-87 EP-A- 0263861 20-04-88 JP-T- 63503062 10-,11-88 DE-A- 3401052 26-07-84 LU-A- SE-A- FR-A,B AU-A- JP-A- GB-A- NL-A- GB-A,B GB-A,B US-A- CH-B- FR-A- BE-A- AU-A- AU-B- 85177 8400254 2539996 2355984 59139390 2137612 8400079 2169598 2169599 4647447 660183 2590484 898708 1018488 574658 24-05-84 22-07-84 03-08-84 26-07-84 10-08-84 10-10-84 16-08-84 16-07-86 16-07-86 03-03-87 31-03-87 29-05-87 16-05-84 28-04-88 14-07-88 EP-A- 0232751 19-08-87 JP-A- 62190175 20-08-87 US-A- 4639365 27-01-87 WO-A- 0602352 24-04-86 AU-A- 4869185 02-05-86 EP-A- 0198051 22-10-86 JP-T- 62501070 30-04-87 WAU-8- 579411 24-11-88 EP-A- 0124766 14-11-84 OE-A- 3316703 08-11-84 s JP-A- US-A- CA-A- 60016936 4719098 1244346 28-01-85 12-01-88 08-11-88 i For mor detrik about this annex,:t Official Journal of the European Patent Office, No. 1/82