WO1998005625A1 - Diagnostic imaging contrast agent with improved in-serum-relaxivity - Google Patents

Abstract

Compounds of formula (I), either in their racemic or enantiomeric forms, wherein R is H or a C1-C20 alkyl chain, which may be interrupted by O, N, S, -CO-, -CH(OH)-, -CH(NH2)-, -CONH-, -NHCO-, -SO-, -SO2-, -SO2NH-, may be substituted by halogen atoms or -COOH groups or their ester or amide derivatives and which is interrupted or not or substituted or not by one or more cyclic R3 residues. R3 is a 5- or 6-membered optionally substituted carbocyclic or heterocyclic, saturated, unsaturated or aromatic cyclic unit, R1, R2 have the same meanings as R, independently from each other, except H, with the proviso that: when R1 and R2 are both C6H5-CH2-O-CH2-, R is different from either H or C6H5-CH2-O-CH2-; as well as the complexes of the compounds of formula (I) with metal ions and the salts thereof with physiologically acceptable organic bases. The compounds are used in the preparation of diagnostic imaging contrast agents and in particular of contrast agents exhibiting improved serum relaxivity.

Description

DIAGNOSTIC IMAGING CONTRAST AGENT WITH IMPROVED IN—SERUM- RE AXIVITY

Technical field of the invention

This invention relates to the Magnetic Resonance Imaging (M.R.I. ), a technique used in the medical diagnosis field for a number of years, to rapidly detect a series of anomalies and/or pathological conditions of living human or animal body organs or tissues, (i. e.: Stark D.D., Bradley W.G. Jr., Eds.: "Magnetic Resonance Imaging", the C.V. Mosby Company, St. Louis, Missouri (USA), 1988). In particular, the invention relates to new chelating agents, especially aminopolycarboxylic acid derivative compounds and to metal chelates thereof with bivalent or trivalent paramagnetic ions and/or salts thereof as well as their use as M.R.I, contrast agents. Background of the invention

Diagnostic imaging techniques, such as Magnetic Resonance Imaging -have been used in medical diagnosis for a long time. The use of contrast media to improve tissue differentiation, to delineate structures or monitor physiological functions constitutes in some cases a fundamental contribution in the best formulation of some medical diagnosis and a valid support for radiologist work.

The medical use of aminopolycarboxylic acid or carboxylic acid derivatives and metal chelates thereof as M.R.I, contrast agents is well known. Said contrast agents, to simplify, can be seen as pertaining to two main groups: the linear and the cyclic ones. The present invention relates to linear polyaminopolycarboxylic acid derivatives, as well as their complexes with paramagnetic metal ions, in particular the Gd³⁺ ion. Patent literature is rich in patent and patent applications relating to the use of linear polyaminopolycarboxylic acid derivatives in the preparation of MRI contrast agents. These compounds generally are derived from the simplest one, N,N,N' ,N* ' ,N* ' -diethylenetnamine-pentaacetic acid, (DTPA), of which the Meglumme salt of the Gd³⁺ complex has been commercialised for a number of years as MAGNEVIST^™) . To improve stability, water solubility and selectivity and to reduce toxicity of these contrast agents generally patent literature proposes the preparation of esters or amide derivatives of said acids or the introduction of substituents on the diethylene unit of the diethylenetriamme DTPA skeleton As an example of said patent literature we can cite: Guerbet EP 661279; Concat Ltd., WO 95/05118; Dibra WO 95/15319; Mallinckrodt WO 94/08630; Green Gross Corp. JP 06016606 and JP 05229998; Mallinckrodt US 5,141,740 and US 5,077,037; Cockbam-Nycomed WO 91/15467 and WO 92/11232; Salutar US 4,889,931 and 4,858,451; Abbot Laboratoires EP 279307; Nyco ed EP 299795; Metasyn Inc. WO 95/28179; Schering EP 680 464; and document cited in these patent publications. Some documents further exist m which substituents have been introduced in (α) to one or more carboxylic DTPA groups; for example: Bracco EP-B-230893 and US 5,182,370; Schering WO 96/16928, WO 96/16929, WO 96/26180 and DE 4341724 enclosing α derivatives, generally comprising an aromatic group, particularly useful for the imaging of the hepatobiliary system. In particular, some patent literature further exists, in which the introduction of an aromatic or lipophilic group on the chelant structure is specifically stated to make the contrast agent as particularly useful for a best definition of the liver and the biliary duct: the

General Hospital Corporation US 4,899,755 and WO -A-

86/06605. summary of the invention

The compounds of the present invention are diethylenetriaminepentaacetic acid derivatives characterised in having substituents at the α position to the carboxy group of two or three of the five acetic groups of DTPA. More precisely, the compounds can have two substituents (the same or different from each other) m α to the carboxyls of the two acetic groups respectively bound to the two side nitrogen atoms of DTPA; or they can have three substituents (the same or different from each, other) in α to the carboxyl groups of three acetic groups respectively bound to the three nitrogen atoms of DTPA.

Therefore, the compounds of the present invention are characterized in having some steπcal hindrance, due to the presence of two or three substituents at the above mentioned positions. The minimum size of the substituents is that of a chain having at least three carbon atoms.

Said hindering groups are probably responsible for the interactions of the paramagnetic chelates with biological components of the fluids in which the agent diffuses, wherein said interactions produce the surprisingly high relaxivity values that we have measured in Human Reconstructed Serum.

Relaxivity values of the contrast agent of the present invention have been tested either in saline or in human serum obtained by Seronorm™ Human, freeze- dried human serum produced by Nycomed Pharma AS, Oslo,

Norway. Serum obtained from said Seronorm™ is substantially equivalent to the fresh one, so its use in the relaxivity determination grants a good picture of the "in vivo" behaviour and, further, an excellent reproducibility of this test.

The compounds object of the present invention are characterised by very high r^ and ^ relaxivity values. When measured in Seronorm™ Human at 20 MHz, at a temperature of 39^*C, and at a concentration comprised from 0 to 1 M, the compounds of the present invention have r_* relaxivity equal to or, preferably, higher than 15 s^-1mM^_1. Detailed disclosure. of the invention

The present invention relates to novel chelating agents, more particularly linear aminopolycarboxylic acid derivatives chelants, and metal chelates thereof and the use of such chelating agents and chelates in the preparation of diagnostic imaging contrast agents and in particular of contrast agents exhibiting improved serum relaxivity.

Said compounds are polyaminopolycarboxylic acid derivatives of formula (I), either in their racemic or enantiomeric forms:

wherein:

R is H or a linear or branched, saturated or unsaturated C,-C '20 alkyl chain, which is interrupted or not by one or more O, N, S atoms or by one or more -CO-, -CH(OH)-, -CH(NH₂)-, -CONH-, -NHC0-, -SO-, -^S02-_' ~S0₂NH-, which is substituted or not with one or more halogen atoms or -COOH groups or their ester or amide derivatives and which is interrupted or not or substituted or not by one or more cyclic R₃ residues which can be the same or different and isolated or fused, with the proviso that, if some of said residues are fused, the maximum number of rings forming the corresponding polycyclic unit is three, in which: is a 5- or 6-membered carbocyclic or heterocyclic , saturated, unsaturated or aromatic cyclic unit, substituted or not with one or more groups X, which can be the same or different, n which: is OH, halogen, NH₂ , NHL, N(L)₂, -O-L, -S-L, -CO-L, where L, the same or different from each other, is C^Cc_j linear or branched alkyl, substituted or not with one or more hydroxy, alkoxy or carboxylic groups, or X is a COOH group or its ester or amide derivative, or a -SO3H group or its amide derivative, and R^ , R₂ have the same meanings as R, independently from each other, except H, with the proviso that: when R^ and R₂ are both CgH₅-CH₂-0-CH₂- , R is different from either H or CgH₅-CH₂-0-CH₂- . The invention further relates to complexes of the ligand of formula (I) with metal ions of atomic number from 20 to 31, 39, from 42 to 44, 49 and from 57 to 83; particularly preferred metals being: Fe(2+)_^ Fe(³⁺),

_Cu(2+) _{/ C}r<³⁺), Gd(³⁺), Eu(³⁺), Dy(³⁺), La<³⁺>, Yb(³⁺), Mn⁽2⁺'; _as well as, where the metal chelate carries an overall charge, a salts thereof with a physiologically acceptable counterion, preferably selected from organic bases such as a primary, secondary or tertiary amines, a basic amino acid, or an inorganic base derived from an alkali metal or alkaline-earth metal cation such as: Na⁺, K⁺, Mg2⁺, Ca²⁺ or a mixture thereof.

The present invention further relates to the use of the compounds of formula (I) and of the salts of the complexes thereof as well as to the pharmaceutical formulations containing them for a diagnostic or therapeutic scope.

Preferred are the compounds of formula (I) in which R, R^ and R₂ are selected from the following groups:

CH,

CH,

^CH>

-CH, -CH. -CH,

CH,

Among the compounds formula (I), particularly preferred are the compounds of formula (II):

(ID where

R₄ = H, or a linear or branched ^C1^~C10 alkyl, optionally interrupted by one or more -CONH- , - NHC0-, -CO- groups and/or N. 0, S atoms, optionally interrupted or substituted with 1 to 3 saturated rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with -OH, -SH, halogen, -COOH, -NH₂ , -N(R")₂, - CON(R")₂, -SO3H, C1-C4 alkoxy groups;

R₅ = independently a linear or branched ^cι~^cιo alkyl, optionally interrupted by one or more -C0NH-, -NHC0- , -CO- groups and/or N, O, S atoms and interrupted or substituted with 1 to 3 saturated rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with -OH, -SH, halogen, -COOH, -NH₂, -N(R")₂, -CON(R")₂, -SO3H, ₁ -C₄ alkoxy groups;

R" = independently H or C_j_-C₅ linear or branched alkyl, optionally substituted with from 1 to 5 -OH groups .

Equally preferred are the compounds of formula (III):

(III) where

H, or a linear or branched ^{c -C}ιo alkyl, optionally interrupted by one or more -CONH-, -NHCO-, -CO- groups and/or N, S atoms and optionally substituted with one or more -OH, -NH₂,

-COOH groups; R₇ = independently a linear or branched

_Q alkyl, optionally interrupted by one or more -CONH-, -NHCO-, -CO- groups and/or N, S atoms and optionally substituted with one or more -OH, -NH₂,

-COOH groups. Equally preferred are the compounds of formula (IV):

(IV) where :

^R '8S H, or linear or branched c₁ c₁₀ alkyl, optionally interrupted by one or more -CONH- , -

NHCO-, -CO- groups and/or N, S atoms, optionally interrupted or substituted with 1 to 3 isolated or fused saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N,

0, S and that are optionally substituted with one or more -OH, -COOH, -NH₂, -N(R")₂, C₁-C₈ alkyl,

C₁-C₆ alkoxy, C₆-C₂₀ arylalkoxy groups; R₉ = independently a linear or branched C^-Cg alkyl, optionally interrupted by one or more

-CONH-, -NHC0-, -CO- groups and/or N, S atoms, which is interrupted or substituted with 2 to 3 fused saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N,

0, S and that are optionally substituted with one or more -OH, -COOH, -NH₂, -N(R") , C₁-Cg alkyl,

C₁-Cg alkoxy, Cg-C_2Q arylalkoxy groups;

R" = independently H or ^cι^_c5 linear or branched alkyl, optionally substituted with 1 to 5 -OH groups. Equally preferred are the compounds of formula (V):

(V) where : ₁₀ = a linear or branched ^ι^_cιo ^al^kγ1' optionally interrupted by one or more -CONH-, -NHCO-, -CO- groups and/or N, S atoms, interrupted or substituted with 1 to 3 saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with one or more -OH, -COOH, -NH₂, - N(R")₂, C₁-C₆ alkyl, C-^-Cg alkoxy groups; ^ = independently a linear or branched C₂-C_1Q alkyl, optionally interrupted by one or more N, S atoms. Two further groups of preferred compounds, all included in compounds of formula (I), are the compounds of formula (VI)

(VI) where : R₁₂ = a linear or branched C₂-C_1Q alkyl, optionally interrupted by one or more -CONH-, -NHCO- , -CO- groups and/or N, S atoms, optionally substituted with one or more -COOH, -NH₂ groups, optionally interrupted or substituted with 1 to 3 saturated, unsaturated or- aromatic, isolated or fused rings, that are optionally interrupted by one or more N, O, S and that are optionally substituted with one or more -OH, -COOH, -NH₂, -N(R")₂, C₁-Cg alkyl, C-,-Cg alkoxy groups, and the compounds of formula(VII)

(VII) where:

^R13 ^{= H}' l^{inear or} branched C^-Cg alkyl, substituted or interrupted with 1 aromatic ring, that is optionally interrupted by one or more N, O, S;

R₁₄ = independently linear or branched C₁-Cg alkyl, substituted or interrupted with one aromatic ring, that is optionally interrupted by one or more N, 0, S. Also preferred among the compounds of the formula

(I), are the compounds of formula (VIII):

(VIII) where ^e15 independently H, halogen;

R₁₆ = H, OH, N(R")₂, COOR", -C0N(R")₂, -S0₃H, -S0₂NHR" ,

C₁-Cg alkyl, C₁-Cg alkoxy; R₁₇ = independently ^cι ~^c alkyl, substituted with

-COOH or -C0N(R")₂ or from 1 to 3 -OH groups; A = direct bond (i.e. no intervening atom), -0- , C=0 = integer 1-6; n = integer 0-2;

R" = independently H or ^cι~ 5 linear or branched alkyl, optionally substituted with 1 to 5 -OH groups with the proviso that, when R^ = H, at least one of the substituents R« c is different from hydrogen.

Particularly preferred, among the various possible synthetic pathways yielding the compounds of the invention, is the following one, which is reported in the following Scheme 1 in order to further clarify the process :

SCHEME 1

OOPg

(2)

natural or synthetic α-amino acid

N-bromosuccinimide

(e) triphenylphosphine

PgO

wherein Pg = protective group (such as t-butyl);

R-, as defined for compounds of general formula (I). Step (a) involves the protection of the alcohol group of 2-bromoethanol with dihydropyran to give intermediate (1). The reaction is carried out in an organic solvent such as CH₂C1₂, CHC1₃, CH₂C1CH₂CL, in the presence of 4-toluenesulfonic acid pyridinium salt or of other acid catalysts. In intermediate (1) the Br atom can be replaced with any other nucleofugal group (such as Cl, I, -OMs , -OTf , -OTs ) and the alcohol- protecting group can be replaced, for example, by benzyl and tπtyl.

In step (b) the ester (for example the t-butyl ester) of a natural or synthetic α-amino acid (2), in the racemic or optically active form, is reacted with intermediate (1) in the presence of diisopropylethylamine in a solvent such as CH3CN, DMF or a chlorinated solvent, to give intermediate (3).

The latter is reacted, in step (c), with a bromoacetic acid ester (such as t-butylbromoacetate ) in the presence of diisopropylethylamine, to give intermediate (4), which is reacted, in the subsequent step (d), with 4-toluenesulfonιc acid pyridinium salt, or other acid catalysts, in a water/ethanol mixture, at a temperature of 20-60^βC, to give intermediate (5).

In step (e), intermediate (5) is brominated with N- bromosuccmimide in the presence of triphenylphosphme, to give compound (6).

With a similar procedure, compound (7) of formula PgOO

is prepared, wherein R is as already defined for compounds of general formula ( I ) .

The Br atom in intermediates (6) and (7) can be replaced with any other nucleofugal group (such as Cl, I, -OMs, -OTf, -OTs).

Intermediates (6) and (7) are then reacted, according to the following Scheme 2, to give the compounds of general formula (I). SCHEME 2

s

compounds of general formula (I wherein R, R., , R₂ are as already defined for compounds of general formula (I).

Step (f) involves the alkylation of the ester of a natural or synthetic α-amino acid (8) with bro oethyl- derivative (6) using double phase conditions in acetonitrile/aqueous phosphate buffer at pH 8 in a 1:1 molar ratio between the two reagents to give compound

(9).

Intermediate (9) is further alkylated with the bromoethyl derivative (7), in step (g), in the same conditions, to give the intermediate pentaester (10), which is deprotected, in step (h), in conventional conditions, to give the corresponding pentaacid. When R-. and R₂ are the same, the dialkylation product (10) can be obtained directly operating in acetonitrile/aqueous phosphate buffer at pH 8 and in a aminoester (8) to bromoderivative (6) molar ratio ranging from 1:2 to 1:3.

An alternative procedure for the preparation of intermediate (6), and similarly of intermediate (7), is illustrated in the following Scheme 3: SCHEME 3

Br

(6) where in :

Pg = protective group (such as t-butyl);

R.1 is above defined for compounds of general formula ( I ) . Step (a¹) involves the condensation of the ester of a natural or synthetic α-amino acid (2) with an α- haloacetic acid ester (2) (such as 2-bromoacetic acid t- butyl ester) in double phase conditions in acetonitrile/aqueous phosphate buffer at pH 8, to give the i inodiacetic acid derivative (2'), which is alkylated, in step (b¹) in 1 , 2-dibromoethane as the solvent, under reflux, in the presence of N,N- diisopropylethylamine and at a temperature of about 80^*C, to give intermediate (6).

Table 1

- continued -

continued -

continued

(*) NaCl 0.15 M in water - pH 7.3 - 20 MHz - 39°C (**) Between 0 and 1 mM ( Seronorm™Human) - 20 MHz

- 39^βC

( *** Between 0 and 2 mM

(§) Bracco EP-B 230893 (^♦) Schering EP 405704

Table 1 above discloses the high relaxivity shown in serum by the compounds of the present invention; r^ and ∑2 relaxivity values of some of the preferred compounds are reported, in comparison with the corresponding r_* and r₂ values measured for some of the mayor prior-art compounds: Gd-DTPA Dimeglumine salt

(MAGNEVIST^(R) ); Gd-BOPTA Dimeglumine salt and Gd-EOB-

DTPA Dimeglumine salt.

The data of Table 1 clearly show that the compounds of the present invention have surprisingly high relaxivity values r^ and r₂, measured in Seronorm™

Human.

This is particularly interesting from the application point of view, both as far as the improvement in the obtainable images, the development of formulations specific to particular districts and the determination of optimum low dosages of the contrast medium are concerned.

EXAMPLE 1 Glvcine 1.1 -d ethvlethvl ester

To a suspension of glycme (22.52 g; 0.3 mol) in t- butyl acetate (1200 L; 9 mol), maintained at 20^βC under an inert atmosphere, HC10, 70% (35 L; 0.41 mol) was added in 1 h. The reaction mixture was stirred at 20°C for 18 h and monitored by TLC. The mixture was extracted with H₂0 (1000 mL ) and the aqueous phase, basified to pH 10 with solid Na₂C0₃ was extracted with CHC1₃ (1800 m ) . The organic layer was dried over Na^SC and concentrated until constant weight to give the desired product (30.4 g; 0.23 mol). Yield 77%.

TLC : Rf 0.5

Stationary phase: silica gel plates 60 ?254

Eluent: 9 : 1 CH₂C1₂/CH₃0H

Detection: 0.5% KMn0₄ in IN NaOH

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure.

EXAMPLE 2

N- 2-Bromoethyl ) -N- r 2- f l . l -dιmethvlethoxv l -2-oxoethvn - L- isoleucme 1 . -dιmethvlethvl ester

A ) L-isoleucine 1 , 1-dιmethylethyles ter

To an ice-bath_, cooled slurry of L-isoleucme (301.0 g; 2.29 mol) in tert-butyl acetate (2.5 L), 70% aq HC10₄ (208 mL, 2.43 mol) was slowly added. The mixture was kept stirring for 10 days at room temperature. Then water (0.5 L) was poured into, followed by cooling in an ice bathand addition of NaOH pellets (100 g) and Na₂C0₃ (110 g) so that pH turned to basic. The mixture was extracted with EtOAc (4 x 0.5 L ) ; the combined organic phases were washed with water (2 x 0.5 L) and brine (0.3 L), at last dried over Na₂S0₄. After careful removal of solvents in vacuo , the desired compound was obtained (262.2 g; 1.40 mol) and stored at -18°C. No further purification was required on the basis of TLC and NMR data. Yield 61%.

TLC: R_f 0.8

Stationary phase: silica gel. Eluent: CH₃Cl/CH₃OH/25% (w/w) NH₄OH 90:9:1.

Detection: 0.2% ninhydπne (w/v) n EtOH.

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

B) ( 2-Bromoethoxy ) (1,1-dιmethylethyl )dιmethylsιlane

Br,

OSιn3uMe₂

This product is commercially available (Aldrich art. 42,842-6).

C) N-[2-(l,l-Dιπ.ethylethoxγ)-2-oxoethyl]-N-[2-[[(l,l- dimethylethyl) dimethylsilyl ]oxy]ethyl 3-L-ιsoleucιne 1 , l-dimethylethyl ester

L-isoleucine 1 , 1-dιmethylethylester (23.00 g; 122.8 mmol ) , (2-Bromoethoxy )-( 1,1-dιmethylethyl )dimethylsllane (30.26 g, 126.5 mmol) and a₂C0₃ (26.18 g; 247.0 mmol) were stirred in DMPU (Aldrich art 25,156-9) (300 L ) at 90°C for 20 h. The intermediate product was not isolated, but after cooling the mixture to roughly 40°C, tert-butyl bromoacetate (commercial product) (19.0 L; 130 mmol) and further Na₂C0₃ (27.00 g; 254.7 mmol) were added, then heating at 90°C was restored. More tert- butyl bromoacetate (2.0 mL; 14 mmol) was added after 4 h and heating prolonged for another 2 h. The mixture was cooled to O'C, then water (600 mL ) was cautiously added

(exothermic solubilization) . Once clear, the solution was extracted with diethyl ether (4 x 250 L ) ; the combined organic layers were washed with water (3 x 250 L ) , brine (250 mL ) , at last dried over Na->S0₄. After removal of solvents in vacuo, the residual oil was purified by flash chromatography (n-hexane/iPr₂0 95:5).

The desired compound was obtained (42.25 g; 91.90 mmol). Yield 75%.

TLC: R 0.75

Stationary phase: silica gel.

Eluent: n-hexane/Et₂0 8:2 (v/v).

Detection: 254 nm; I₂; 0.5% KMn0₄ in 1 N NaOH; 2%

(w/v) Ce(S0₄)₂-4H₂0, 4.2% (w/v) ( NH₄ ) ₆Mo₇0₂₄ , 6% (w/v) ^H2^S04 ^{in ater} (Pancaldi).

-^•■H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

D) N-[2-(l,l-Dimethylethoxy)-2-oxoethyl]-N-( 2-hydro- xyethyl)-L-isoleucine 1 , l-dimethylethyl ester

In a solution of the compound obtained in the previous step (33.57 g; 73.02 mmol) in freshly dried THF

(200 mL, distilled over sodium/benzophenone ) cooled at

-10"C under a nitrogen atmosphere, a 1 M solution of nBu₄NF in THF (110 mL; 110 mmol) was slowly dropped. The stirred solution was allowed to rise to room temperature throughout 5 h. The solvent was then removed on a rotavapor, the residue taken up in diethyl ether (400 L), and the solution washed with water (100 mL ) , saturated NH₄HC0₃ (200 mL ) , water (100 mL ) , brine (100 mL ) . After prolonged evaporation in vacuo (250 Pa) a thick oil was obtained (28.71 g): the crude product was deemed pure enough for the following derivatization . TLC: R_f 0.5 Stationary phase: silica gel. Eluent: n-hexane/EtOAc 85:15 ( w/v ) .

Detection: 254 nm; I₂; 0.5% KMn0₄ in 1 N NaOH; 1% (w/v) vanilline in 96% (w/v) H₂S0₄/abs EtOH 4:1 (v/v). ¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure. E) N-(2-Bromoethyl)-N-[2-(l,l-dimethylethoxy )-2-oxo- ethyl]-L-isoleucine 1 , l-dimethylethyl ester

To a solution of the crude product obtained from the previous preparation (73 mmol approximately) and triphenylphosphine (20.31 g; 77.43 mmol) in CH₂C1₂ (0.5 L, freshly distilled over CaH₂ ) under a nitrogen atmosphere, N-bromosuccinimide (>98%; 13.80 g) was portionwise added throughout 1 h at 0"C. The reaction was left stirring overnight and allowed to rise gradually to room temperature. Most of the solvent was evaporated, diethyl ether and n-hexane were added in sequence, enabling the bulk of Ph₃P0 to precipitate as a filterable solid. The remaining solution was concentrated in the presence of silica gel and submitted to a flash chromatography ( n-hexane/iPr₂0 91:9). The desired compound was isolated (21.22 g; 51.96 mmol).

Yield over the last two steps: 71%.

TLC: R_f 0.5

Stationary phase: silica gel.

Eluent: n-hexane/iPr₂0 9:1 (v/v).

Detection: 254 nm; I₂; 0.5% KMn0₄ in 1 N NaOH.

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

EXAMPLE 3

N-(2-Bromoethyl)-N-[2-(l,l-dιmethylethoxy)-2-oxoethylj-

_L-phenylalamne 1 , l-dimethylethyl ester

A) L-phenylalamnβ 1 ,1-dιmethylethyl ester

98 % H₂S0₄ (7 L; 0.13 mol) was dripped over 20 min into dioxane (70 mL ) , maintaining the temperature of the solution below 20^βC. After addition of L-phenylalanme (commercial product) (16.5 g; 0.10 mol), the solution was stirred over 12 h at 132 kPa under an isobutene (commercial product) atmosphere (consumed isobutene 45 g; 0.80 mol). The solution was dropped into a mixture of ice (200 g) and 10 N NaOH (30 mL , 0.30 mol) and extracted with Et₂0 (1 L). After washing with H₂0 (150 L), the organic phase was dried over Na₂S0₄ and evaporated in vacuo. The residue was distilled to give L-phenylalanine 1 , l-dimethylethyl ester (13 g;

0.059 mol) . Yield 59 %. bp : 85 - 90^βC at 5.3 Pa

Acidic titer (0.1 N HCl) : 99.7 %; equivalent point pH 4.77

HPLC : 98 % (area %) - Chromatographic method:

Stationary phase: Lichrosorb RP-Select B 5 (?)m;

250 x 4 mm column packed by Merck KGaA;

Temperature: 45⁰C; Mobile phase: gradient elution;

A = 0.01 M KH₂P0₄ and 0.017 M H₃P0₄ in water

B = CH₃CN

Gradient timetable: mm % A % B

0 95 5 30 20 80

45 20 80

Flow rate: 1 mL mm^-1;

Detection (UV): 210 nm , 280 nm;

Injection: 10 μL; Sample concentration: 1 g mL^-1;

Instrumentation : Merck KGaA - Hitachi high pressure gradient pump system (two Lachrom L 7100 pumps), Merck

KGaA - Hitachi Lachrom L 7200 autosampler, Merck KGaA

- Hitachi Lachrom L 7300 column thermostat, Merck KGaA - Hitachi Lachrom L 7400 UV detector.

K.F. : < 0.1%

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

-°V 20^U + 16.64°(c 5.29, CHC1. Elemental analysis (%):

C H N

Calcd. 70.56 8.65 6.33

Found 71.21 8.89 6.61 B) N-[2-(l,l-Dιmethylethoxy)-2-oxoethyl]-N-(2-hydro- xyethyl )-L-phenylalanιne 1 , 1-dιmethylethγl ester

A solution of L-phenylalanine 1 , l-dimethylethyl ester (221.3 g; 1 mol), 2- ( 2-bromoethoxy ) tetrahydro- pyran, prepared according to J. Org. Che . 1986, 51, 752-755 (282.3 g; 1.35 mol) and diisopropylethylamine (commercial product) (175 mL; 1 mol) in CH₃CN (1 L) was refluxed for 14 h. Diisopropylethylamine (commercial product) (175 L; 1 mol) and tert-butyl bromoacetate (commercial product) (233 g; 1.2 mol) were added and the mixture refluxed for further 2 h. The solution was evaporated to give a residue which was dissolved in n- hexane (2 L) and washed with H₂0 (1,4 L), 1 N HCl (500 L), 1 N NaOH (100 mL ) and H₂0 (200 mL ) . The solution was evaporated and the residue was dissolved in MeOH (2 L) and 2 N HCl (1 L was added. After 2 h, 2 N NaOH (1.2 L) was added, the solution was evaporated to remove methanol and n-hexane (2 L) was added to extract the product. The organic solution was evaporated to obtain the desired product (280 g; 0.738 mol). The product was utilised for the following step without further purification. Yield 74 %.

HPLC : 91 % (area %) - Chromatographic method of previous step A). [α]_D ²⁰: + 17.13° (c 5.08, CHC1₃ )

In another preparation the compound was purified by flash chromatography :

Stationary phase: Silica gel 230-400 mesh Merck KGaA art 9385

Eluent: 4 : 1 n-hexane/EtOAc to give N-[2- ( 1 , 1-dimethylethoxy )-2-oxoethyl]-N- ( 2- hydroxyethyl )-L-phenγlalanine 1 , l-dimethylethyl ester for the analytical characterisation.

Acidic titer (0.1 N HC10₄) : 98.6 %

HPLC : 97.4 % (area %) Chromatographic method of previous step A).

K.F. : < 0.10%

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

[α]_D ²⁰: - 19.89" (c 5.01, CHC1₃ ) Elemental analysis

C H N

Calcd. 66.46 8.76 3.69

Found 66.13 9.32 3.68

C ) N-{ 2-Bromoethyl )-N-[2- ( 1 , 1-dimethylethoxy )-2-oxo- ethyl]-L-phenylalanine 1 , l-dimethylethyl ester

N-Bromosuccinimide (46.3 g; 0.26 mol) was added in portions to a solution of the product obtained in the previous step (75.9 g; 0.20 mol) and triphenylphosphine

(68.1 g; 0.26 mol) in CH₂C1₂ (500 mL ) cooled at 0 5"C and stirred. The solution was allowed to rise to r.t. and, after 4 h, was washed with H₂0 (400 mL ) , 5 % aq. NaHC0₃ (200 mL ) and H₂0 (100 L ) . After drying

(Na₂S0₄) the solution was evaporated and the residue was suspended in Et₂0 (1 L); the solid (triphenylphosphine oxide) was filtered and the solution evaporated. The residue was dissolved in n-hexane (500 L ) , Carbopuron 4N (commercial product) (4 g) was added and filtered after stirring for a while. The solution was evaporated to give a residue (77 g) that was purified by flash chro atography (Stationary phase: Silica gel 230-400 mesh Merck KGaA art. 9385 (1 kg); Eluent: Et₂0) to afford the desired compound (68 g; 0.154 mol). Yield

77%.

TLC : Rf 0.46

Stationary phase : Silica gel plates 60 ^254 (Merck

KGaA code 5715)

Eluent: n-hexane/EtOAc 4 : 1

Detection : 1 % KMn0₄ in IN NaOH

HPLC : 92 % (area %) - Chromatographic method of previous step A).

EXAMPLE 4

N-(2-Bromoethyl)-N-[2-(l,l-dimethylethoxy)-2-oxoethyl]-

L-tryptophan 1 , l-dimethylethyl ester

A) N -[ (Phenylmethoxy )carbonyl]-L-tryptophan

To a suspension of tryptophan (20.08 g; 98.32 mmol) in H₂0, cooled to 0^βC, was added IN NaOH (98 L ; 98 mmol) and the solution became clear. Maintaining the temperature at 0°C, IN NaOH (108 L; 108 mmol) and benzyl chloroformate (CBZC1, commercial product) (15.38 mL; 107.7 mmol) were simultaneously dropped in the reaction mixture. After the addition the mixture was kept at 0°C for 30 mm and then allowed to rise to r . t.

The reaction was monitored by HPLC (Chromatographic method of Example 3, A). After further 3 h the pH was corrected to 9.5 by addition of IN NaOH and the mixture was washed with Et₂0 (200 L ) . The aqueous layer was acidified to pH 2 using 2N HCl and the precipitate was filtered through a G3 septum, washed with cold water (2 x 200 L ) and dried over P₂°5 under vacuum (2 kPa ) . N- [ (Phenylmethoxy)carbonyl]-L-tryptophan (33.96 g) was obtained and used in the following step without further purification. .p. : 230^*C

TLC: Rf 0.60 Stationary phase: silica gel plates 60 F₂5₄

Eluent 9 : 1 : 0.1 CHCl₃/MeOH/AcOH

Detection: 254 n ; 0.5% KMn0₄ in IN NaOH

HPLC: 95.6% Chromatographic method of Example 3, A).

¹³C-NMR, ^H-NMR and MS spectra were consistent with the structure.

B) N-[ ( Phenylmethoxy )carbonyl ] -L-tryptophan 1, l-dimethylethyl ester

Into a suspension of N- [( phenylmethoxy ) carbonyl ]-L- tryptophan (33.27 g; 98.32 mmol), benzyltrlethylammonium chloride (BTEAC) (22.4 g; 98.32 mmol) and K₂C0₃ (176.91 g; 1.28 mol) in dimethylacetamide (750 L ) , tert-butyl bromide (265 mL; 2.36 mol) was dropped. The solution was heated to 55°C and maintained under vigorous stirring for 19 h. The reaction was monitored by HPLC

(Chromatographic method of Example 3, A)). The solution was cooled to r.t., diluted with H₂0 (3 L) and then extracted with EtOAc (2 L). The organic layer was washed with H₂0 (2 L) and, after elimination of the solvent, N-

[ (phenylmethoxy ) carbony1] -L-tryptophan 1 , 1-dimethylethyl ester (36 g) was obtained and used in the following step without further purification.

TLC: Rf 0.44

Stationary phase: silica gel plates 60 F₂₅₄

Eluent 7 : 3 n-hexane/EtOAc

Detection: 254 nm; 0.5% KMn0₄ in IN NaOH

HPLC: 99 % (area %) Chromatographic method of Example 3,

A). ¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure .

C) L-Tryptophan 1 , l-dimethylethyl ester

To a solution of N- [ (phenylmethoxy )carbonyl ]-L- tryptophan 1 , l-dimethylethyl ester (36 g; 98 mmol) in EtOH (150 mL) Pd/C 10% (5 g) was added. A H₂ atmosphere was set and hydrogenation was performed with the aid of a Venturi type stirrer. Reaction conversion was estimated by HPLC (Chromatographic method L/46). After 6 h at r.t. the suspension was filtered through paper, then through Millipσre(^R) HA 0.45 μm and the filtrate was concentrated under reduced pressure to give a crude oil. The crude was dissolved in CHC1₃ (200 mL ) and washed with 5% aq. Na₂C0₃ (200 mL ) and brine (150 mL ) . The organic layer was dried over Na₂S0₄ and concentrated to give L-tryptophan-l,l-dιmethylethyl ester (18.16 g), which was used in the following step without further purification. HPLC: 97 % (area %) Chromatographic method of Example 3,

A).

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure .

D) N-[2-(l ,1-Dιmethylethoxy )-2-oxoethy1] -L-tryptophan 1 ,1-dιmethylethyl ester

To a solution of L-tryptophan 1 , l-dimethylethyl ester (17.10 g; 65.68 mmol) in CH₃CN (150 mL ) 2M phosphate buffer ( pH 8; 150 mL ) was added. Tert-butyl bromoacetate (10.7 mL; 72.25 mmol) was dropped into the mixture and vigorous mechanical stirring set on. The reaction was monitored by HPLC (Chromatographic method of Example 3, A)).. After 23 h the organic layer v/as separated and concentrated to dryness to give a crude oil (26.62 g), which was purified by flash chromatography (silica gel; n-hexane/ethyl acetate, 8:2 v/v) to give the desired product (20.07 g; 53.59 mmol). Yield 54.5% starting from L-tryptophan. TLC: Rf 0.28

Stationary phase: silica gel plates 60 F₂^₄ Eluent 7 : 3 n-hexane/EtOAc Detection: 254 nm; 0.5% KMn0₄ in IN NaOH HPLC: 100 % (area %) Chromatographic method of Example 3, A). ¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure .

E) N-[2-(l ,l-Dιmethylethoxy)-2-oxoethyl]-N-(2-hydro- xyethyl )-L-tryptophan 1 , l-dimethylethyl ester

In a four-necked flask cooled to -80"C equipped with a mechanical stirrer, a thermometer, and a jacketed dropping funnel N-[2-( 1 , 1-dιmethylethoxy )-2-oxoethyl ]-L- tryptophan 1 , l-dimethylethyl ester (5 g; 13.35 mmol) was dissolved in CH₃CN (25 L ) . In the dropping funnel, cooled at -80°C, ethylene oxide (13 mL; 0.26 mol) was collected from the cylinder and then quickly dropped into the solution. Solid ytterbium tπflate (0.83 g; 1.34 mmol) was added and after removal of the cooling bath the temperature was allowed to rise to r.t. The reaction was monitored by HPLC (Chromatographic method of Example 3, A)). After 15 h the solution was diluted with H₂0 (50 L) and extracted with Et₂0 (150 L ) . After evaporation of the solvent the crude was purified by flash chromatography (silica gel; n-hexane/ethyl acetate, 7:3 v/v) giving the desired product (4.32 g; 10.32 mmol) . Yield 77 %. TLC: Rf 0.23 Stationary phase: silica gel plates 60 F₂54 Eluent 7 : 3 n-hexane/EtOAc

Detection: 254 nm; 0.5% KMn0₄ in IN NaOH HPLC: 99 % (area %) Chromatographic method of Example 3,

A).

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure . F) N-(2-Bromoethyl)-N-[2-(l,l-dιmethylethoxy)-2-oxo- ethyl]-L-tryptophan 1 , l-dimethylethyl ester

To a solution of N- [ 2- ( 1 , 1-dιmethylethoxγ )-2- oxoethyl ]-N-( 2-hydroxyethyl ) -L-tryptophan 1 , 1-dιmethylethyl ester (4.64 g; 11.09 mmol) in CH₂C1₂ (44 mL ; freshly distilled over CaH₂ ) under an inert atmosphere, solid Ph₃P (2.9 g; 11.09 mmol) was added. The solution was cooled to 0°C and then solid NBS (1.97 g; 11.09 mmol) was portionwise added (45 mm), waiting for complete dissolution after each addition The reaction was monitored by TLC: 1. Stationary phase: silica gel plates 60 F₂₅₄ Eluent 7 : 3 n-hexane/EtOAc Detection: 254 nm; 0.5% KMn0₄ in IN NaOH 2. Stationary phase: silica gel plates 60 F₂^₄ Eluent 8 : 2 n-hexane/EtOAc Detection: 254 nm; 0.5% KMn0₄ in IN NaOH

After 3 h at 0°C and 1 h at r.t. the mixture was concentrated until a white solid started to precipitate and the cloudy solution was left on standing at 4°C for 72 h. The white precipitate (Ph₃P0) was filtered off and the clear solution concentrated. The crude was purified by flash chro atography (silica gel; n-hexane/ethyl acetate, 8:2 v/v) to give the desired product (4.46 g;

9.26 mmol) . Yield 83 %.

TLC: Rf 0.42

Stationary phase: silica gel plates 60 F₂₅₄

Eluent 8 : 2 n-hexane/EtOAc

Detection: 254 nm; 0.5% KMn0₄ in IN NaOH

HPLC: 94 % (area %) Chromatographic method of Example 3,

A).

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure .

EXAMPLE 5

N-(2-Bromoethyl)-N-[2-(l,l-dimethylethoxy)-2-oxoethyl]-

0-(phenylmethyl )-L-senne 1 , l-dimethylethyl ester

A) 2-( 2-Bromoethoxy ) tetrahydropyran This compound, has been prepared according to: J. Org. Che . 1986, 51, 752-755.

B) O-Phenylmethyl-L-serine 1 , 1-dimethylethylester

To a suspension of O-phenylmethyl-L-serine (50 g; 0.26 mol) in t-butyl acetate (1000 mL; 7.49 mol) 70% perchloric acid was added (50 mL; 0.58 mol). The solution was maintained under stirring for 72 h at 25°C under inert atmosphere. The reaction mixture was diluted with Et₂0 (300 mL), then a 10% Na₂C0₃ aqueous solution was slowly added until pH 9 was reached. The organic layer was separated, dried over Na₂S0₄ and concentrated. The residual t-butyl acetate was distilled under reduced pressure (40°C; 0.1 mm Hg ) . The desired product was obtained (54.2 g; 0.21 mol). Yield 83%.

TLC: Rf 0.4

Stationary phase: silica gel Eluent: CHC1₃ : CH₃OH = 9.5 : 0.5 (v/v)

¹³C-NMR, ^H-NMR and MS spectra were consistent with the structure .

[α]_D ²⁰: -9,08° (c 5.0; CHC1₃)

C) N-(2-Hydroxyethyl)-N-[2-(l,l-dimethylethoxy)-2-oxo- ethyl]-0-(phenylmethyl )-L-serine 1 , l-dimethylethyl ester

A solution of O-phenylmethyl-L-serine 1,1- dimethylethylester (251 g; 1 mol), 2-(2-bromo- ethoxy ) tetrahydropyran (244 g; 1.1 mol) and diisopropylethylamine (commercial product) (155 g; 1.2 mol) in CH₃CN (1 L) was refluxed for 14 h. Diisopropylethylamine (commercial product) (193 g; 1.5 mol) and tert-butyl bromoacetate (234 g; 1.2 mol) were added and the mixture refluxed for further 2 h. The solution was evaporated and the residue was dissolved in CH₂C1₂ (2 L) and washed with H₂0 (3 L). The solution was evaporated and the residue (650 g) dissolved in 90% EtOH (2 L); 4-toluenesulfonic acid pyridinium salt (obtained by salification of 4-toluenesulfonic acid with pyridine in diethyl ether, filtration of the precipitate and drying) (30 g; 0.12 mol) was added and the solution heated at 55°C for 45 h. The solution was evaporated, the residue dissolved CH₂C1₂ (2 L) and the solution washed with H₂0 (1 L), 5% Na₂C0₃ (1 L) and H₂0 (1 L).

The organic solution was dried on Na₂S0₄ and evaporated; the residue (450 g) was purified by flash chromatography :

Stationary phase: Silica gel 230-400 mesh Merck KGaA art

9385

Eluent: 4:1 n-hexane/EtOAc

The desired product was obtained (172 g; 0.42 mol). Yield 42 %. mp : 31°C

Acidic titer (0.1 N HC10₄) : 98.0 %

TLC: Rf 0.32

Stationary phase: Silica gel plates 60 ₂^₄ (Merck KGaA code 5715)

Eluent: 8 : 2 n-hexane/EtOAc

Detection: 1% KMn0₄ in IN NaOH

HPLC: 96 % (area %) Chromatographic method of Example 3,

A). [α]_D ²⁰: + 0.46° (c 5.6, CHC1₃)

-*-³C-NMR, "--H-NMR, MS and IR spectra were consistent with the structure.

Elemental analysis (%):

C H N Calcd. 64.52 8.61 3.42

Found 65.70 9.01 3.33

D) N-( 2-Bromoethyl )-N-[2-( 1 , 1-dimethylethoxy ) -2-oxo- ethyl]-0-(phenylmethyl )-L-serme 1 , l-dimethylethyl ester

N-Bromosuccmimide (commercial product) (13.9 g; 0.078 mol) was added in portions to a solution of N-(2- hydroxyethyl )-N-[2-( 1 , 1-dιmethylethoxy ) -2-oxoethyl ] -O- (phenylmethyl )-L-serme 1 , l-dimethylethyl ester and triphenylphosphine (commercial product) (20.5 g; 0.078 mol) in CH₂C1₂ (250 L ) cooled at 0 - 5°C and stirred. After 4 h the solution was washed with H₂0 (100 L ) , 5% NaHC0₃ (100 mL) and H₂0 (100 mL ) After drying (Na₂S0₄) the solution was evaporated and the residue was suspended in Et₂0 (150 mL ) , the solid (triphenylphosphine oxide) was filtered and the solution evaporated. The residue (31 g) was purified by flash chro atography : Stationary phase: Silica gel 230-400 mesh Merck KGaA art 9385 (200 g)

Eluent: 9:1 n-hexane/EtOAc

The desired product was obtained (24 g; 0.051 mol).

Yield 78%.

Potentiometric titer (0.1 N HCIO₄/CH3COOH ) : 101 % Argentometric titer (0.1 N AgN0₃ after demolition with KOH/DMSO): 101.2% TLC: Rf 0.40

Stationary phase: Silica gel plates 60 F₂₅₄ (Merck KGaA code 5715) Eluent: n-hexane/EtOAc 9 : 1

Detection: 1% KMn0₄ m IN NaOH GC: 98 % (area %) - Gaschromatographic method:

Stationary phase: CIP-SIL DB 5; Film thickness: 0.25 μm; Column (WCOT): 10 m x 0.53 mm; Carrier (He) flow rates: column flow rate: 10 mL mm^-1; split flow rate: 110 mL mm--^*-; make up flow rate: 30 mL mm--^*-; septum purge flow rate: 4 L min^"--; Detector (FID) feeding: hydrogen pressure: 1.2 bar; air pressure. 2.8 bar; Oven temperature timetable : initial temperature: 150°C initial time: 2 mm rate: 20°C mm^-1 final temperature: 210°C final time: 25 mm; Injector temperature: 250°C; Detector temperature: 250°C; Injection: 3 μL; Sample concentration: 25 mg mL- ;

Instrumentation: Hewlett - Packard HP 5890. [α]_D ²⁰: - 2.51° (c 4.2, CHCl₃)

¹³C-NMR, ^-H-NMR, MS and IR spectra were consistent with the structure. Elemental analysis (%):

C H Br N Calcd. 55.93 7.26 16.91 2.97 Found 55.96 7.26 16.76 2.93 EXAMPLE 6 [Compound 1] [ [ [-N,N'-[ ( Carboxymethyl ιmιno)dι-2,l-ethanediyl ] bis [ N- carboxymethyl-L-isoleucmate] 1(5-) ]gadolιnate( 2- ) ] disodium salt

A) N-( 2-Bromoethyl)-N-[2-(l , 1-dimethylethoxy ) -2-oxo- ethyl]-L-ιsoleucιne 1 , l-dimethylethyl ester

The product is prepared according to Example 2.

B) Glycme 1 , l-dimethylethyl ester

H_jN^^COOtBu

The product is prepared according to Example 1.

C) N,N'-[[[2-(l ,1-Dimethylethoxy )-2-oxoethyl ] l lno]dl- 2 , 1-ethanedιγl ]bιs [N- [2-( 1 ,1-dimethylethoxy )-2-oxo- ethyl]L-ιsoleucιne 1 , l-dimethylethyl ester]

To a solution of N- ( 2-bromoethyl ) -N- [ 2- ( 1 , 1- dimethylethoxy )-2-oxoethyl ]-L-ιsoleucιne 1 , 1-dimethylethyl ester (8.16 g; 25 mmol) and glycine 1,1- dimethylethyl ester (1.31 g; 12.5 mmol) in CH₃CN (100 mL ) , maintained under vigorous stirring, 2M phosphate buffer pH 8 (100 L ) was added. The biphasic mixture was stirred at 20°C for 48 h. The organic layer was separated and the solvent evaporated on a rotavapor. The residue was taken up in CH C1₂ (100 L ) and the solution washed with water (100 mL ) and brine (100 mL ) . The organic layer was dried over a₂S0₄, concentrated and the residue purified by flash chromatography (n- hexane/EtOAc 9:1 v/v) to give the desired compound (8.2 g; 12.5 mmol). Yield 83.5%.

TLC : Rf 0.5

Stationary phase: silica gel plates 60 F₂c₄

Eluent: 8 : 2 n-hexane/EtOAc

Detection: 254 nm; 0.5% KMn0₄ in IN NaOH

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure.

K.F. : < 0.1 % Elemental analysis (%): C H N

Calcd. 64.17 10.13 5.35 Found 64.01 10.57 5.20

D) N,N' -[ (Carboxymethylimino)dι-2 , 1-ethanediyl ]bis [N- carboxymethyl-L-isoleucine]

To a solution of the pentaester of the previous preparation (7.92 g; 10.1 mmol) in CHC1₃ (150 mL ) maintained at 0-5°C under an inert atmosphere, (CH₃)₃SiI (13.6 mL; 0.1 mol) was added in 30 m . The solution was stirred for 4 days at 20°C, following the reaction by HPLC (chromatographic method of Example 3, A)). The reaction mixture was cooled to 5°C and H₂0 (150 mL ) was added. After separation, the pH of the aqueous phase was adjusted to pH 1.7 with ION NaOH and the solution was loaded onto a column of Amberlιte(^R) XAD 1600 resin (500 mL), which was eluted with H₂0 (5 L) and then with

H₂0/CH₃CN (gradient elution 90:10 —>75:25 v/v ratios). After evaporation of the solvent the desired product was obtained (4.3 g; 8.5 mmol). Yield 84%. p: 117-120°C

HPLC : 99.9% (area %)

1. Chromatographic method of Example 3, A). 2. Chromatographic method:

Stationary phase: Spheri -10 RP-2 10 μm;

250 x 4,6 mm column packed by Applied Biosystem;

Temperature: 50"C;

Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-octylamme is added to 240 mL of acetonitrile mixed with 760 L of water The solution is buffered to pH 6 with H₃P0₄;

Flow rate: 1.0 L min^-1,

Detection (UV): 200 nm; Injection: - 10 μL;

Sample concentration: 2 mg mL

Instrumentation: Hewlett - Packard HP 1090 M liquid chromatograph equipped with DR 5 solvent delivery system, autosampler, column thermostat and diode array detector.

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent v/ith the structure.

K.F. : 0.44 % Elemental analysis (%): C H N Calcd. 52.27 7.78 8.31 Found 52.53 7.93 8.78 anhydrous

E) [ C [N,N*-[ (Carboxymethylimino)di-2,l-ethanediyl]bis- [N-carboxymethyl-L-isoleucinate] ] ( 5- ) ]gadolinate( 2- ) ] disodiu salt

A suspension of the free ligand from the previous preparation (2.53 g; 5 mmol) in H₂0 (70 mL ) at 5°C was neutralized by addition of IN NaOH. To the clear solution was slowly added a 0.2 M solution of GdCl₃ (25 L; 5 mmol) maintaining the mixture at pH around 7 by addition of IN NaOH. The resulting cloudy solution was stirred for 30 min at room temperature, then filtered over Millipore GSWP 0,22 m. The clear solution was loaded onto a column of Amberlite XAD 1600 polystyrene resin (300 mL ) , eluted with H₂0 (2 L) and then with H₂0/CH₃OH (1 L; 90:10 v/v). The title product (3.4 g; 4.83 mmol) was obtained. Yield 97%. mp : > 300°C

Free ligand (0.001 M GdCl₃) : < 0.1% HPLC : 100% (area %)

1. Chromatographic method of Example 3, A).

2. Chromatographic method 2 of previous step D).

MS and IR spectra were consistent with the structure.

K.F. : 7.80%

Weight loss (130°C) : 7.72% Elemental analysis (%):

C H N Gd Na calcd. 37.55 4.87 5.97 22.34 6.53 found 37.36 4.98 5.89 22.20 6.56

EXAMPLE 7 [Compound 2]

[[[1S-[1R*(1R*,2R*) , 2R*]]-N, N-Bis[ 2- [(carboxymethyl ) (1- carboxy-2-methylbutyl )amino]ethyl]-L-isoleucinate-

( 5- ) ]gadolinate(2-) ] disodium salt.

A) N- (2-Bromoethyl)-N-[2- (1,1-dimethylethoxy )-2-oxo- ethyl]-L-isoleucine 1 , l-dimethylethyl ester

The product is prepared according to Example 2. B) [1S-[1R*(1R*,2R*) , 2R* ] ]-N , -Bis [2- [ [ 1- [ ( 1 , 1-dime- thylethoxy )carbonyl]-2-methγlbutyl] [2-[ ( 1 , 1-dimethyl- ethoxy)-2-oxoethyl]amino]ethyl]-L-isoleucine 1 , l-dimethylethyl ester

An emulsion of L-isoleucine tert-butyl ester (Example 2, Step A) (1.89 g; 10.1 mmol) and of N-(2- Bromoethyl )-N-[2-(l , 1-dimethylethoxy )-2-oxoethyl]-L-iso- leucine 1 , l-dimethylethyl ester (8.97 g; 22.0 mmol) in acetonitrile (75 L ) and 2 M pH 8 phosphate buffer (50 L ) was vigorously stirred at room temperature for 3 days; the aqueous layer was substituted with fresh buffer and stirring went on for another day. As reaction rate sensibly decreased when conversion of starting materials approached 70 to 80%, conditions were forced by heating at 50°C for 40 h and at 70°C for 16 h. The phases were allowed to separate and cool; the organic layer was evaporated and taken up in EtOAc, the aqueous layer was extracted with EtOAc (2 x 100 mL ) . The combined organic layers were washed with water (2 x 150 L ) , brine (100 mL ) and at last dried over Na₂S0₄. The crude (11.74 g) was purified by flash chro atography ( n- hexane/iPr₂0 9:1 to 8:2). After careful removal of solvents in vacuo, the desired compound was obtained

(5.55 g; 6.59 mmol). Yield 65%.

TLC: R_f 0.4

Stationary phase: silica gel. Eluent:

9:1 (v/v). Detection: 254 nm; I₂;0.5% KMn0₄ in 1 N NaOH. ¹H-NMR , ¹³C-NMR, MS and IR spectra were consistent with the structure.

C) [1S-[1R*(1R*,2R*) , 2R*]-N,N-Bis[ 2- [(carboxymethyl )- ( 1-carboxy-2-methylbutyl )amino]ethyl]-L-isoleucine

To a solution of the pentaester obtained in the previous preparation (6.36 g; 7.55 mmol) in CHC1₃ (0.3 L, freshly distilled over CaH₂) under a nitrogen atmosphere, lodotnmethylsilane (12.0 mL; 88.2 mmol) was slowly added at -15°C. The mixture was allowed to gradually rise to room temperature and left stirring for 3 days. Then it was cooled in an ice bath and so much 1 N NaOH was added that a pH value of 10 could be established in the upper layer. Vigorous stirring was prolonged until both phases became homogeneous and separable. After separation the organic phase was extracted with 0.1 N NaOH (100 mL ) ; the combined aqueous layers were washed with diethyl ether (2 x 200 mL ) , then concentrated to a volume of 100 m . To the solution warmed in a steam bath at 40°C, 9 N HCl was slowly added under vigorous stirring; as pH drifted under a value of 5, acidification was brought forth even slower with 2 N HCl down to a pH value of 2.74 (below which precipitation of a white unworkable gum began) The just acidified solution was loaded onto a column of resin Amberlιte(^R' XAD 1600 (370 m ) . After initial very slow percolation of water (which drew off not only inorganic salts but also a minor quantity of the desired product as a polysodium salt), a gradient elution of water/ace- tomtrile was applied (95:5, 0.5 L; 90 10, 0.5 L; 85:15 0.5 L; 80:20, 0.5 L; 75:25 0.5 L, 70 30, 0.5 L; 65:35, 1 L). The homogeneous fractions were combined and concentrated to a volume of 500 L This solution [containing 3.3 mmol of ligand] was submitted to the ensuing complexation without isolation of the product. Approximate yield- 44%. mp: broad softening range (125-175'C)

HPLC: 100% (area) - Chromatographic method: Stationary phase: Lichrospher 100 RP-8 5 μm;

250 x 4 mm column packed by Merck KGaA; Temperature: 45°C;

Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-octylamme is added to 262 mL of acetonitrile mixed with 738 mL of water. The solution is buffered to pH 6.0 with H₃P0₄; Flow rate: 1.3 mL mm^-1;

Detection ( UV ) : 205 nm; Injection: 30 μL;

Sample concentration: 1 or 5 g mL^-1;

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (L6200 and L6000), Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T 6300 column thermostat, Merck KGaA - Hitachi L 4250 UV detector. ¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

D) [[[1S-[1R*(1R*,2R*) , 2R* ]] -N , N-Bis [ 2- [( carboxymethyl ) ( l-carboxy-2-methylbutyl ) amino] ethyl ]-L-ιsoleu- cinatef 5- ) ]gadolmate( 2- ) ] disodium salt.

To the aqueous solution (500 L ) of the ligand obtained in the previous preparation (ca. 3.3 mmol) Gd₂0₃ (596 mg; 1.64 mmol) and 0.1 N NaOH (64 mL; 6.4 mmol) were added. The reaction progress was monitored by HPLC. After setting on heating at 60°C for 3 h, the mixture became clear although complexation was not complete, so another portion of Gd₂0₃ (64 mg; 0.18 mmol) was added and stirring maintained for 4 days at room temperature. Because at this point a relevant quantity of free ligand was again discernible in the HPLC pattern, although some oxide was available in the mixture, further large excess d₂0₃ (427 g; 1.18 mmol) was added and heating restored at 65°C for 14 h.

Complete conversion was achieved by addition of Gd(0Ac)₃

(91 mg; 0.22 mmol) and brief heating at 65°C. The slurry was then cooled to room temperature, filtered through paper and concentrated on a rotavapor. Azeotropic distillations with toluene were repeated in order to remove any trace of acetic acid. The residue was diluted in water/ ethanol 95:5 (100 mL ) and loaded onto a column of resin Amberlιte(^R) XAD 1600 (280 mL ) , conditioned with water/methanol 95:5. A gradient elution of water/methanol was applied (95:5, 92:8, 86:14 - percentage at which the chelate complex began to elute - 82:18, 78:22, 74:26, 70:30; 7 x 0.5 L). After removal of solvent-s in vacuo and repeated azeotropic distillations with toluene, the title compound was obtained (2.41 g; 3.17 mmol). Approximate yield: 96%. Yield over the last two steps: 42%. p: > 295°C (dec) HPLC: 97% (area) - Chromatographic method:

Stationary phase- Lichrospher 100 RP-8 5 μm;

250 x 4 mm column packed by Merck KGaA; Temperature- 45°C; Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-octylamme is added to 325 mL of acetonitrile mixed with 675 mL of water. The solution is buffered to pH 6.0 with H₃P0₄;

Flow rate: 1.3 mL min^-1;

Detection ( UV ) : 205 nm;

Injection: 30 μL; Sample concentration: 5 mg mL^-1;

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (L6200 and L6000), Merck KGaA

- Hitachi AS 2000 autosampler, Merck KGaA T 6300 column thermostat, Merck KGaA - Hitachi L 4250 UV detector. MS and IR spectra were consistent with the structure.

K.F. : 5.17%

Specific rotation (305): Cα]₄₃₆ ²⁰ = + 1.7°; [α]₄₀₅ ²⁰ = +

5.7°; [α]₃₆₅ ²⁰ = + 11.5°; (c 1.16; CH₃OH)

Weight loss (130°C): 6.32% Elemental analysis (after drying at 130°C) (%):

C H Gd N Na

Calcd. 41.10 5.57 20.69 5.53 6.05

Found 41.08 5.78 20.46 5.48 6.02

EXAMPLE 8 [Compound 3] [ [ [-N,N'-[ (Carboxymethylιmιno)dι-2 , 1-ethanedιyl ]bιs [N- carboxymethyl-L-tryptophanate] ] ( 5-) ]gadolιnate( 2-) ] disodium salt

A) Glycine 1 , l-dimethylethyl ester The product is prepared according to Example 1.

B) N-(2-bromoethγl)-N-[2-(l,l-dιmethylethoxy)-2-oxo- ethyl] -L-tryptophan 1 , l-dimethylethyl ester

The product is prepared according to Example 4. C) N,N'-[ [ [2-( 1,1-Dimethylethoxy )-2-oxoethyl ] lmmo]dl- 2 , l-ethanediyl]bιs[N-[ 2-( 1 , 1-dimethylethoxy )-2-oxo- ethyl]L-tryptophan 1 , l-dimethylethyl ester]

To a solution of glycine 1 , l-dimethylethyl ester (1.7 g; 12.5 mmol) and N- ( 2-bromoethyl )-N-[ 2-( 1 , 1- dimethylethoxy )-2-oxoethyl ] -L-tryptophan 1,1-di methyl- ethyl ester (12 g; 24.9 mmol) CH3CN (70 L ) , 2M phosphate buffer pH 8 (100 mL ) was added. The bifasic mixture was maintained under vigorous mechanical stirring for 20 h. The organic layer was separated and concentrated. The oily residue was purified by flash chromatography ( n-hexane/EtOAc 8:2) to give the desired compound (17.5 g; 18.75 mmol). Yield 75%. TLC : Rf 0.45

Stationary phase: silica gel plates 60 F₂^₄ Eluent: 7 : 3 n-hexane/EtOAc Detection: 254 nm; 0.5% KMn0₄ in IN NaOH HPLC : 98.9% (area %) - Chromatographic method of Example 3, A) .

¹³C-NMR, ¹H-NMR, MS and IR spectra weie consistent with the structure. K.F. : 0.56% Elemental analysis (%):

D ) N , N ' - [ ( Carboxymethylιmιno )dι-2 , l-ethanedιyl ] bιs [ N- car box methyl- L-tryptophan ]

To a solution of the pentaester obtained in the previous preparation (8.51 g; 9 mmol) in anhydrous CHC1₃ (150 L ) maintained at 0-5°C under an inert atmosphere, (CH₃)₃SιI (12.4 L; 90 mmol) was added in 30 mm. The temperature was allowed to rise to r.t., the violet solution was stirred for 64 h and the reaction progress was monitored by HPLC (Chromatographic method of Example 3, A)). After cooling to 0°C the reaction mixture was maintained under vigorous stirring, while IN NaOH (120 mL ) was added to achieve complete dissolution of the precipitate The organic layer was separated and the aqueous phase was acidified to pH 3 with 6N HCl. The precipitate was filtered off, washed with cold H₂0 (200 mL ) , and dried over P₂Oc until constant weight (6 g) The brownish solid was suspended H₂0 (100 mL ) and 3N HCl was added until complete dissolution (pH 1.5) The solution was loaded onto a column filled with Amberlιte-^R) XAD 1600 resin (600 L ) which was eluted with H₂0 (1000 mL) and then with H₂0/CH₃CN (gradient elution 90:10—>85:15 v/v ratios). After evaporation of the solvent the desired compound (4 g; 6.14 mmol) was obtained. Yield 68%. mp: 168-170°C

HPLC : 99.5% (area %) - Chromatographic method of

Example 3 , A) .

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure.

K.F. : 2.28%

Elemental analysis (%):

C H N

Calcd. 58.98 5.72 10.75

Found 58.55 5.79 10.73 anhydrous

E) [ [ [-N,N'-[ (Carboxymethylιmιno)dι-2,l-ethanedιyl]- bιs[N-carboxymethyl-L-tryptophanate] ] ( 5- ) ]gadolinate-

(2-)] disodium salt

To a solution -of the free ligand from the previous preparation (3.23 g; 5 mmol) in H₂0 (100 mL ) brought to pH 6.5 with IN NaOH, Gd₂0₃ (0.91 g; 2.5 mmol) was added and the suspension was warmed at 70°C for 5 h. The reaction course was monitored by HPLC: Chromatographic method:

Stationary phase: Lichrospher 100 RP-8 5 (?)m;

250 x 4 mm column packed by Merck KGaA; Temperature: 40°C; Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-octylamine is added to 300 L of acetonitrile mixed with 700 mL of water. The solution is buffered to pH 6 with H₃P0₄;

Flow rate: 1 mL m ^-1;

Detection (UV): 200 nm;

Injection: 10 μL; Sample concentration: 1 mg mL^-1;

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (L6200 and L6000), Merck KGaA

- Hitachi AS 2000 autosampler, Merck KGaA T 6300 column thermostat, Merck KGaA - Hitachi L 4250 UV detector. The cloudy solution was filtered through a

Millipore (^R ) HA 0.45 filter and the pH was adjusted to

6.7 with IN NaOH. The filtrate was concentrated under reduced pressure to give the title compound (3.3 g; 3.88 mmol) . Yield 78%. mp : > 250°C

Free ligand (0.001 M GdCl₃) : < 0.1%

HPLC : 99.8% (area %) - Chromatographic method of above.

MS and IR spectra were consistent with the structure.

K.F. : 14.09% Elemental analysis ( % ) :

C H N Gd Na calcd. 45.23 3.80 8.24 18.50 5.41 found 45.36 3.69 8.20 18.26 5.17 anhydrous

EXAMPLE 9 [Compound 4] [[[[S-(R*,R* )]-N,N'-[[(l-Carboxy-2-methylbutγl)ιmιno]dι-

2, l-ethanedιyl]bιs[N-carboxymethyl-L-tryptophanate] ]-

( 5- ) ]gadolmate( 2- ) ] disodiu salt

A) L-isoleucine 1 , l-dimethylethyl ester

The product is prepared according to Example 2, Step A. B) N-(2-bromoethyl)-N-[2-(l , 1-dιmethylethoxy )-2-oxo- ethyl]L-tryptophan 1 , l-dimethylethyl ester The product is prepared according to Example 4. C) [S-(R*, *)]-N,N'-[[l-[( 1,1-Dime hylethoxy) car- bonyl]-2-methylbutyl]imino]di-2 , 1-e anediyl ]bis [N- [ 2- ( 1 ,ldimethylethoxy)-2-oxoethyl ] -L-tryptophan 1, l-dimethylethyl ester]

To a solution of L-isoleucine 1 , 1-dimethγlethyl ester (0.86 g; 4.59 mmol) and N- ( 2-bromoethyl ) -N- [ 2- ( 1 , 1-dimethylethoxy )-2-oxoethyl] -L-tryptophan 1 , 1-dime- thylethyl ester (4.42 g; 9.18 mmol) in CH₃CN (65 mL ) 2M pH 8 phosphate buffer (65 L ) was added. The reaction was maintained under vigorous mechanical stirring and followed by T.L.C.. After 3 h the aqueous phase was replaced with the same amount of fresh 2M pH 8 phosphate buffer and the same operation was repeated after 18 h.

After 23 h the organic layer was separated and evaporated. The residue was purified by flash chromatography : 1^st column: silica gel; n-hexane/ethyl acetate, 8:2 v/v

2^st column: silica gel, CHCl₃/MeOH, 15:0.2 v/v

The desired product (3.88 g; 3.9 mmol) was obtained.

Yield 85.5 %.

TLC: Rf 0.25 Stationary phase: silica gel plates 60 F₂₅₄

Eluent 8:2 n-hexane/EtOAc

Detection: 254 nm; 0.5% KMn0₄ m IN NaOH

¹³C-NMR, ^-H-NMR and MS spectra were consistent with the structure. D) [S-(R*,R*)]-N,N'-[[(l-Carboxy-2-methylbutyl)ιmmo]- dι-2 , lethanedιyl]bιs[N-carboxymethyl-L-tryptophan]

Into a solution of the pentaester from the previous preparation (6.0 g; 6.07 mmol) in CH₂C1₂ (freshly distilled over CaH₂ ) in an inert atmosphere (CH₃)₃SιI (8.3 L; 60.97 mmol) was slowly dropped (0.5 h) maintaining the temperature around 0°C. After removal of the cooling bath the temperature was allowed to rise to r.t and the reaction was followed by HPLC (Chromatographic method of Example 3, A)). After 44 h fresh (CH₃)₃SιI (5 mL; 36.73 mmol) was added and after further 70 h (overall 114 h) other (CH₃)₃SιI (1 mL; 7.35 mmol) was added. After 23 h (overall 137 h) the solution was poured into a 250 L becker and vigorously stirred while IN NaOH (5 x 100 mL ) was added. The mixture was settled after each addition of NaOH until the organic layer became clear and from HPLC analysis the desired product completely disappeared. After separation the aqueous layer was acidified to pH 6.5 by addition of 37%

HCl and the solution, without isolation of the product, used for the following complexation . p: 185 °C dec.

HPLC: 95% (area %) Chromatographic method of Example 3,

A).

K.F. : 7.15%

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure .

Elemental analysis (%):

C H N Cl Na

Calcd. 61.09 6.41- 9.89

Found 60.91 6.08 10.19 — — anhydrous

E) [[[[S-(R*,R*)]-N,N'-[[(l-Carboxy-2-methylbutyl)- ιmmo ] dι-2 , l-ethanedιyl ]bιε [ N-carboxymethyl-L-trypto- phanate] ] ( 5- ) ]gadolinate( 2- ) ] disodium salt

To the solution coming from the previous step were simultaneously added a solution of GdCl₃ ^• 6 H₂0 (2.26 g; 6.07 mmol) in H₂0 (50 mL ) and IN NaOH to maintain the mixture at pH 6.5. The reaction was followed by HPLC analysis. After 1 h the pH was adjusted to 7 with IN

NaOH and the solution loaded onto a column of

Amberlιte(^R) XAD-1600 resin (500 L ) . The column was eluted with H₂0 until complete elimination of the salts, then the product was eluted with a solution of H₂0/CH₃CN 9:1. The solution containing the product (90% purity) was eluted a second time through Amberlιte(^R) XAD-1600 resin (500 mL ) m a similar way. A portion of the product was not pure enough yet, so it was necessary a third purification through Amberlιte'^R) XAD-1600 resin (500 mL ) but this time the resin was conditioned and the product was dissolved in H₂0/CH₃CN 9:1. The solution containing the purified product (pH 8.7) was concentrated to 50 mL and, while maintained under stirring, some Dowex CCR-3 LB resin was added until the pH of the solution^, was adjusted to 4 The resin was filtered through a G3 septum and washed with warm (40°C) H₂0 (100 L). The pH of the clear solution was corrected to 7 by addition of IN NaOH. After elimination of the solvent the title compound (2.18 g; 2.41 mmol) was obtained. Yield 40% starting from the pentaester mp- > 250°C

HPLC: 100% (area %) - Chromatographic method: Stationary phase- Lichrospher 100 RP-8 5 (⁹)m, 250 x 4 mm column packed by Merck KGaA; Temperature: 45°C;

Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-nonylamine is added to 330 mL of acetonitrile mixed with 670 mL of water. The solution is buffered to pH 6 with H₃P0₄;

Flow rate: 1 L min -1. Detection ( UV ) : 245 nm; Injection: 10 μL;

Sample concentration:! mg mL^-1;

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (L6200 and L6000), Merck KGaA

- Hitachi AS 2000 autosampler, Merck KGaA T 6300 column thermostat, Merck KGaA - Hitachi L 4250 UV detector.

K.F.: 12.91%

MS and IR spectra were consistent with the structure.

Elemental analysis (%):

C H N Gd Na Calcd. 47.73 4.45 7.73 17.36 5.08 Found 47.19 4.28 7.67 17.12 5.13 anhydrous EXAMPLE 10 [Compound 5]

[[[1S-[1R*(1R*,2R*) ,2R*]]-N,N-Bιs[ 2- [(carboxymethyl) (1- carboxy-2-methylbutyl ) amino] ethyl ] -L-tyros inate- ( 5- ) ]gadolinate( 2- ) ] disodiu salt

A) N-(2-Bromoethyl)-N-[2-(l , 1-dimethylethoxy )-2-oxo- ethyl]-L-isoleucine 1 , l-dimethylethyl ester

The product is prepared according to Example 2.

B) L-tyrosine tert-butyl ester

This product is commercially available (Novabioche art 04-12-5026, batch no. A09451 - CAS No. [16874-12-7]).

C) [1S-[1R*(1R*,2R*) ,2R*]]-N,N-Bis[2-[[l-[(l,l-dime- thylethoxy)carbonyl]-2-methylbutyl] [2-{ 1 ,1-dimethyl- ethoxy )-2-oxoeth 1] amino]ethyl] -L-tyrosine 1 , l-dimethylethyl ester

An emulsion of• L-tyrosine tert-butyl ester (2.15 g; 9.06 mmol), N- ( 2-bromoethyl )-N-[ 2- ( 1 , 1-dimethylethoxy )- 2-oxoethyl]-L-iεoleucine 1,1-dimethyl ethyl ester (7.43 g; 18.2 mmol) in acetonitrile (150 L ) and 2 M phosphate buffer pH 8 (100 mL ) was vigorously stirred at room temperature for 2 days; the aqueous layer was substituted with fresh buffer and stirring went on for another day. As reaction rate sensibly decreased when conversion of starting materials approached 70 to 80%, a slight excess of N-( 2-bromoethyl )-N-[2- ( 1 , 1-dimethyl- ethoxy )-2-oxoethyl]-L-isoleucine 1,1-dimethyl ethyl ester (1.12 g; 2.74 mmol) was added ai stirring was prolonged 4 days more. The phases were allowed to separate; the organic layer was evaporated and taken up in EtOAc, the aqueous layer was extracted with EtOAc

(300 L). The combined organic layers were washed with water (400 mL ) , brine (100 L ) and at last dried over

Na₂S0₄. The crude (11.74 g) was purified by flash chromatography ( n-hexane/EtOAc 9:1 to 8:2). After careful removal of solvents in vacuo, the desired compound was obtained (8.13 g; 9.11 mmol). Quantitative yield.

TLC: R_f 0.4

Stationary phase: silica gel.

Eluent: n-hexane/EtOAc 75:25 (v/v).

Detection: 254 nm; I₂; 0.5% KMn0₄ in 1 N NaOH. ^-H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

D) [1S-[1R*(1R*,2R*) , 2R* ] ]-N , N-Bis [2- [ ( carboxymethyl )- ( 1-carboxy-2-methy1-butyl)amino]ethyl] -L-tyrosine

To a solution of the pentaester obtained from the previous preparation (7.63 g; 8.55 mmol) in CHC1₃ (250 mL, freshly distilled over CaH ) under a nitrogen atmosphere, iodotri ethylsilane (14.0 L; 102 mmol) was slowly added at -15°C. The mixture was allowed to gradually rise to room temperature and then was stirred for 3 days. Then it was cooled in an ice bath and so much 1 N NaOH was added that a pH value of 10 could be established in the upper layer. Vigorous stirring was prolonged until both phases became homogeneous and separable. After separation the organic phase was extracted with 0.1 N NaOH (100 L ) ; the combined aqueous layers were washed with diethyl ether (400 mL ) , then concentrated to a volume of 100 L . To the solution warmed in a steam bath at 50°C, 6 N HCl was slowly added under vigorous stirring; as pH drifted under a value of 5, acidification was brought forth even slower with 2 N HCl down to a pH value of 2.70 (below which precipitation of a white unworkable gum began). The just acidified solution was loaded onto a column of resin Amberlite(^R) XAD 1600 (250 L ) . After initial very slow percolation of water, a gradient elution of water/acetonitrile was applied (95:5, 0.5 L; 92.5:7.5, 0.5 L; 90:10 0.5 L; 87:13, 0.5 L; 84:16, 0.5 L ; 80:20, 1 L; 76:24, 0.5 L; 72:28, 0.5 L; 68:32, 0.5 L; 64:36, 0.5 L; 60:40, 0.5 L). The homogeneous fractions were combined and concentrated to a volume of 1000 mL; this solution was submitted to the ensuing complexation without isolation of the ligand. Approximate yield: 50%. mp: broad softening range (115-175°C), then decomposition

HPLC: 98.2% (area) - Chromatographic method: Stationary phase: Lichrospher 100 RP-8 5 μm;

250 x 4 mm column packed by Merck KGaA; Temperature: 45°C; Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-octylamine is added to 244 mL of acetonitrile mixed with 756 L of water. The solution is buffered to pH 6.0 with H₃P0₄;

Flow rate: 1.3 mL min ,-^"1

Detection (UV): 210 nm;

Injection: 10 μL;

Sample concentration: 1 mg L -^"1.

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (L6200 and L6000), Merck KGaA

- Hitachi AS 2000 autosampler, Merck KGaA T 6300 column thermostat, Merck KGaA - Hitachi L 4250 UV detector.

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

Specific rotation : [α]_D ²⁰ = -41.2°; (c 1.13; DMF )

K.F. : 2.66%

Elemental analysis (%):

C H Calcd. 56.94 7.41 Found 57.12 7.56

E) [[[1S-[1R*(1R* ,2R*) , 2R* ] ]-N ,N-Bis [ 2- [( carboxymethyl ) ( 1-carboxy-2-methylbutyl) amino] ethyl ]-L-tyrosinate- ( 5- ) ]gadolinate( 2- ) ] disodium salt

To the aqueous solution (750 mL ) of the free ligand [ca. 3.29 mmol from the previous preparation] Gd₂0₃ (604 mg; 1.67 mmol) and 0.1 N NaOH (65.8 mL; 6.58 mmol) were added. The reaction progress was monitored by HPLC

(Chromatographic method of the previous step E)). After setting on heating at 60°C for 3 h, the mixture became clear although complexation was not complete, so another portion of Gd 0₃ (23 mg; 0.063 mmol) was added and stirring maintained for 4 days at room temperature. The slurry was then filtered through paper and concentrated on a rotavapor to 100 mL . The resulting clear solution was slowly percolated (flow 40 mL/h) through a column of Doweχ(^R) CCR3LB (Na⁺ form; 35 mL ) . The collected eluates underwent liophilization and the title compound was isolated (2.42 g; 2.99 mmol).

Approximate yield: 91%. Yield over the last two steps: 45%. p: > 240°C (dec)

HPLC: 100% (area) - Chromatographic method: Stationary phase: Lichrospher 100 RP-8 5 (?)m;

250 x 4 mm column packed by Merck KGaA; Temperature: 45 °C; Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-octylamine is added to 298 mL of acetonitrile mixed with 702 mL of water. The solution is buffered to pH 6.0 with H₃P0₄; Flow rate: 1.3 L min^-1; Detection ( UV ) : 210 nm;

Injection: 10 μL;

Sample concentration: 1 and 5 mg mL^-1; Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (L6200 and L6000), Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T 6300 column thermostat, Merck KGaA - Hitachi L 4250 UV detector. MS and IR spectra were consistent with the structure.

Specific rotation (300): [α]₅₈₉ ²⁰ = -20.8' [α] 20 _

578

-21.4; Cα]₅₄₆ ²⁰ = -29.8°; [α]₄₃₆ ²⁰ = -46.9' [α] 20 _ 405^'

-54.2°; [α]₃₆₅ ²⁰ = -72.1° (c 1.11; CH₃OH)

K.F.: 8.57%

Weight loss (130°C): 8.82%

Elemental analysis (%):

C H Gd N Na Calcd. 43.01 4.98 19.42 5.19 5.68 Found 42.96 5.19 18.69 5.04 5.59 EXAMPLE 11 [Compound 6]

[[[4S-[4R*,8(R*) ,12R*]]-4-Carboxy-8-[l-carboxγ-2-(4- hγdroxyphenyl)ethyl]-5 , ll-bis( carboxymethyl )-l-phenyl- 12- [ (phenylmethoxy ) methyl ]-2-oxa-5, 8 , 11-triazatridecan- 13-oate( 5- ) ]gadolinate( 2- ) ] dihydrogen compound with 1 deoxy-1-methylamino-D-glucitol (1:2)

A) L-tyrosine 1 , l-dimethylethyl ester

This product is commercially available (Novabiochem art. 04-12-5026 - CAS No. [16874-12-7]).

B) N-(2-Bromoethyl)-N-[2-(l , 1-dimethylethoxy )-2-oxo- ethyl]-0-(phenylmethyl )-L-serine The product has been prepared according to Example 5.

C) [4S-[4R*,8(R* ) ,12R*]]-4-[(l,l-Dimethylethoxγ)carbo- nyl]8-[l-[(l, 1-dimethylethoxy )carbonyl]-2- ( 4-hydroxyphe- nyl)ethyl]-5 , ll-bis[2-( 1 , 1-dimethylethoxy )-2-oxoethy1]1- phenyl-12-[ ( phenylmethoxy ) methyl ] 2-oxa- 5 ,8,11- tπazatridecan-13-oic acid 1 , l-dimethylethyl ester

L-Tyrosine 1 , 1-dιmethylethy 1 ester (5.1 g ; 22 mmol) was added to a stirred solution of N- ( 2-bromoethyl ) -N- [2-(l , 1-dimethylethoxy )-2-oxo hy1 ]-0- ( phenylmethyl )-L- serine 1 , l-dimethylethyl ester (21.6 g ; 46 mmol) in CH₃CN (250 mL ) . A 2 M pH 8 phosphate buffer solution (350 mL ) was added and the resulting biphasic mixture was vigorously stirred for 12 h. The two phases were separated and fresh 2 M pH 8 phosphate buffer solution (200 L ) was added to the organic phase. After stirring for an additional 2 h the organic phase was evaporated under reduced pressure (2 kPa ) and the residue dissolved in CH₂C1₂ (300 mL ) . The resulting solution was washed with water (200 mL ) , dried over Na-,S0₄ and concentrated to residue. The crude (28.7 g) was purified by flash chromatography:

Stationary phase: silica gel 230 - 400 Mesh (E. Merck art. 9385)

Eluent: 8 : 2 to 6 : 4 hexane/EtOAc

The desired product was obtained (19.9 g, 19 mmol). Yield 90%.

HPLC: 96.5 % (area %) Chromatographic method of Example 3 , A ) .

TLC : Rf = 0 . 26

Silica gel plates 60 F₂₅₄ (E. Merck art. 5715)

Eluent: 8 : 2 hexane/EtOAc Detection: UV (254 nm) and 1% KMn0₄ in 1 M NaOH

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure .

D) [4S-[4R*,8(R*) ,12R*]]-4-Carboxy-8-[l-carboxy-2-(4- hydroxyphenyl ) ethyl] -5 , ll-bιs( carboxymethyl )-l-phenyl- 12 [ ( phenylmethoxy )methyl]-2-oxa-5 ,8 , 11-trιazatrιdecan-

13-oιc acid

CF₃COOH (7 mL; 10.4 g; 90 mmol) was added to a solution of [4S-[4R*,8(R*),12R*]]-4-[{l,l-dιmethyletho- xy )carbonyl]-8-[l-[ (1,1-dimethyl ethoxy )carbonyl ]-2- ( 4- hydroxyphenyl ) ethyl] -5 ,ll-bis[-2-(l, 1-dimethylethoxy )-2- oxoethyl]-l-phenyl-12-[ (phenylmethoxy ) methyl]-2-oxa- 5,8,ll-triazatridecan-13-oιc acid 1 , l-dimethylethyl ester (22.4 g; 22 mmol) in CH₂C1₂ (100 L ) cooling th mixture at 0 - 5°C. The result σ solution was evaporated under reduced pressure (2 kPa ) and the residue was dissolved n CfgCOOH (150 L; 224 g; 2 mol). The solution was stirred at room temperature for 60 h, then evaporated (2 kPa) and the residue was washed with CH-_jCl (3 x 150 L ) evaporating each time the solvent under reduced pressure (2 kPa ) . The resulting crystalline material was suspended in diethyl ether (100 mL ) and filtered. After repeating the treatment for three times the crude material (17 g) was dissolved m a

1:1 EtOH/H₂0 mixture and loaded onto an Amberlιte(^R) XAD 1600 polystyrene resm (700 mL; conditioned with 1:1

EtOH/H₂0) eluting with 1:1 EtOH/H₂0 to give the desired product (14. mmol). Yield 64%.

HPLC: 96 % (area %)

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure.

[α]_D ²⁰ -8.20° (c 1.025, 0.4 M NaOH) Elemental analysis (%):

C H N calcd. 60.06 6.14 5.68 found 59.37 6.33 5.46

E) [[[4S-[4R*,8(R* ) ,12R*]]-4-Carboxy-8-[l-carboxy-2- ( 4-hydroxyphenyl ) ethyl] -5 , 11-bis ( carboxymethyl )-l-phe- nyl-12-[ ( phenylmethoxy )methyl ]-2-oxa-5 ,8,11-trιazatπ- decan-13-oate( 5- ) ]gadolιnate( 2- ) ] dihydrogen compound with 1-deoxy-l-methylammo-D-glucιtol (1 2)

A 0.875 M solution of 1-deoxy-l-methylammo-D- glucitol (commercial product) (48.4 L; 42.3 mmol) was dropped into a suspension of [4S- [ 4R* , 8 ( R* ) , 12R* ] ] -4- carboxy-8-[l-carboxy-2-( 4-hyd oxyphenyl )ethyl]-5,ll- bis (carboxymethyl ) -l-phenyl-12- [ ( phenylmethoxy ) methyl ]-

2-oxa-5 ,8 , 11-trιaza tπdecan-13-oιc acid (14 mmol) m H₂0 (100 m ) , stirring until complete dissolution A 0 477 M solution of GdCl₃ (29 5 mL ; 14 mmol) was slowly added maintaining the mixture at pH around 7 by addition of a 0.875 M solution of 1-deoxy-l-methylammo-D- glucitol (31.4 L; 27.4^" mmol). The resulting cloudy solution was stirred for 30 mm at room temperature, then filtered over Mιllιpore(^R> (HA-0,22 μ ) . A IM solution of HCl (28 mL; 28 mmol) was added to the clear solution under stirring until pH 2.0 was reached. The resulting precipitate was filtered, washed with water (5 x 40 mL ) and then suspended in water (100 m ) . The pH of the suspension was adjusted to neutrality by addition of a 0.875 M solution of 1-deoxy-l-methylammo-D-glucιtol

(25 mL; 22 mmol) stirring until complete dissolution of the acid complex. The neutral solution was evaporated (2 kPa) and the residue dried to give the title compound

(15.2 g;11.8 mmol). Yield 84%. m. p. : 151'C

HPLC: 94.5 % (area %) - Chromatographic method: Stationary phase: E. Merck Lichrospher 100 RP-18 5 μm;

250 x 4 mm column packed by E.Merck;

Temperature: 50°C;

Mobile phase- isocratic elution with premixed mobile phase: 1 g of n-octylamme s added to 350 mL of acetonitrile mixed with 650 mL of water The solution is buffered to pH 6 with H₃P0₄;

Flow rate: 1 mL min^-1;

Detection ( UV ) : 210 nm;

Injection: 10 μL; Sample concentration: 1 mg mL^-1;

Instrumentation: E. Merck - Hitachi L 6000 isocratic pump, E. Meick - Hitachi AS 2000 autosampler, Rheodyne

7414 six-port injection valve, E. Merck T 6300 column thermostat, E. Merck - Hitachi L 4250 UV detector MS and IP spectra were consistent with the structure Elemental analysis (%):

C H N Gd calcd. 47.68 5.97 5.54 12.24 found 47.85 6.03 5.46 12.11

EXAMPLE 12 [Compound 7]

[[ [4S-[4R*,8(1R*,2R*) , 12R* ]] -4-Carboxy- , 11-bis ( carboxymethyl )-8-[ ( 1-carboxy-2-methyl ) butyl ]-l-phenyl-12- [ (phenylmethoxy ) methyl ]2-oxa- 5 ,8 , 1 l-tπazatridecan-13- oate( 5- ) ]gadolinate( 2- ) ] dihydrogen compound with 1- deoxy-l-( ethylamino ) -D-glucitol (1:2)

A) L-Isoleucine 1 , 1-dιme hylethy 1 ester

The product has been prepared according to Example 2, Step A.

B) N-(2-Bromoethγl )-N-[2-( 1 , 1 -di ethylethoxy ) -2-oxo- ethyl]-0-(phenylmethyl )-L-se ine 1 , 1-d imethylethyl ester

The product has been prepared according to Example 5.

C) [4S-[4R*,8(lR*,2R*),12F*]]-4-[(l , 1 -Dimethylethoxy ) - carbonyl]-8-[ [l-[ ( 1 , 1-dimethylethoxy ) carbony1] -2- methyl]butyl]-5,ll-bis[2-( 1 , 1-dιmet hylethoxy )-2-oxo- ethyl]-l-phenyl-12-[ ( phenylme hoxy ) methyl ] -2-oxa-5 , 8 , 11- triazatridecan-13-oic acid 1 , l-dimethylethyl ester

A 2 M pH 8 phosphate buffer (1710 mL ) was added to a solution of N- ( 2-bromoethyl )-N- [ 2- ( 1 , 1-dιmethyl- ethoxy )-2-oxoethyl]-0-( phenyl ethyl )-L-seπne 1,1-dιme- thylethyl ester (181.69 g; 0.36 mol) and L-isoleucme 1,1-dιmethylethyl ester (34.48 g; 0.17 mol) m CH₃CN (1430 mL). After 23 h of vigorous stirring the two phases were separated and further 2 M pH 8 phosphate buffer (850 mL ) was added to the organic phase. After 23 h the organic phase was separated and evaporated under reduced pressure (2 kPa ) . The residue was dissolved in (1700 mL ) and the resulting solution was washed with water (850 L ) , dried (Na S0₄) and concentrated to dryness (2 kPa). T-he crude (174.8 g) was purified by flash chromatography (Stationary phase: silica gel 230 - 400 mesh (1250 g); Eluent: 9:1 4:1 hexane/EtOAc gradient) to give the desired compound (150.6 g, 0.15 mol) . Yield 91%. HPLC: 92.5 % (area %) - Chromatographic method:

Stationary phase: Merck KGaA Lichrosorb RP-Select B 5 μm;

250 x 4 mm column packed by Merck KGaA; Temperature: 35°C; Mobile phase: gradient elution; A = 0.017 M H₃P0₄ in water Gradient timetable: mm % A % B

Flow rate: 1 mL m ^-1;

Detection ( UV ) : 210 nm;

Injection: 10 μL;

Sample concentration: 1 mg mL^""1;

Instrumentation : Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck

KGaA - Hitachi L 3000 diode array detector.

TLC: Rf = 0.38

Silica gel plates 60 F₂₅₄ (Merck KGaA art. 5715) Eluent: 4 : 1 hexane/EtOAc

Detection: UV (254 nm ) and 1% KMn0₄ in 1 M NaOH

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure.

K.F. : 0.33 % Elemental analysis { % ) :

C H N calcd. 66.84 9.06 4.33 found 66.14 8.97 4.25

D) [4S-[4R*,8(1R*,2R*) , 12R* ] ]-4-Carboxy-5 , 11-bιs ( car- boxymethyl )-8-[ ( l-carboxγ-2-methyl ) butyl ]-l-pheny1-12-

[ (phenylmethoxy )methyl]-2-oxa-5,8,ll-trιazatπdecan-13- oic acid

CF₃COOH (35 9 mL; 53.4 g, 0 47 mol) was added to a solution of the pentaester from the previous preparation (124 g; 0.128 mmol) in CH₂C1₂ (540 L ) maintaining the temperature at 0 — 5°C. The resulting solution was evaporated (2 kPa ) and the residue was dissolved in CF₃COOH (1070 mL; 1600 g; 14.04 mol). The solution was stirred at room temperature for 69 h and then evaporated to dryness (2 kPa). The crude (215.8 g) was dissolved in a 1-1 MeOH/H₂0 mixture (670 ) and loaded onto a column of Amberlite XAD 1600 polystyrene resin (1.9 L). After elution with 7/3 MeOH/H₂0 the crude ligand was obtained (71 4 g) The product was dissolved in H₂0 (300 mL ) and the pH adjusted to. 11 with 10 N NaOH (42.15 mL; 0.42 mol) The solution was maintained at pH 11 for 16 h by the slow addition of 10 N NaOH (6.26 L ; 62.6 mmol) through a pH-stat apparatus Acidification of the mixture to pH 2 with 1 N HCl led to the formation of a precipitate which was filtered, washed with H 0 and dried to give the desired compound (63 g; 91.3 mmol). Yield 72% m p 108°-110° C Acidic titer (0.1 N NaOH) : 99.7% Complexometric titer (0.1 N ZnS0₄ ) ^• 96.7%

HPLC 96.0% (area %) - Chromatographic method: Stationary phase: Lichrosorb RP-Select B 5 μm;

250 x 4 mm column packed by Merck KGaA;

Temperature: 35°C;

Mobile phase: isocratic elution: A/B = 55:45 A = 0.017 M H₃P04 water

B = CH₃CN

Flow rate: 1 mL m n^-1;

Detection ( UV ) : 210 nm;

Injection: 10 μL; Sample concentration: 1 mg mL^-1;

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (two Lachrom L 7100 pumps), Merck

KGaA - Hitachi Lachrom L 7200 autosampler, Merck KGaA

- Hitachi Lachrom L 7300 column thermostat, Merck KGaA - Hitachi Lachrom L 7400 UV detector.

K.F. : 1.80 %

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure.

[α]_D ²⁰: -26.03° (c 2.01, 0.4 N NaOH) Elemental analysis {%):

C H N calcd. 59.21 6.87 6.09 found 59.68 6.80 6.15 anhydrous

E) [[[4S-[4R*,8(1R*,2R*) , 12R* ] ] -4-Carboxy- 5 , 11-bis- caroxymethyl)-8-[ ( 1-carboxy-2-methyl )buty 1 ] -l-phenyl-12-

[ (phenyl-methoxy ) methyl ]-2-oxa- 5 ,8 , ll-trιazatridecan-13- oate( 5- ) ]gadolιnate( 2- ) ] dihydrogen compound with 1- deoxy-l-( methylamino )-D-glucιtol (1:2)

A I M aqueous solution of 1-deoxy-l- ( methylamino )- D-glucitol (120 L; 1^' mmol) was dropped into a suspension of the frt ligand from the previous preparation (27.6 g; 40 mmol) in H₂0 (200 mL ) , stirring until a clear solution was obtained. A solution of GdCl₃ • 6 H₂0 (14.9 g; 40 mmol) m H₂0 (50 mL ) was slowly added, maintaining the mixture at pH 7 by addition of a 1 N aqueous solution of 1-deoxy-l- ( methylamino )-D-glucιtol (75.28 L; 75.28 mmol) by means of a pH-stat apparatus. The reaction mixture was filtered through a Millipore HA 0.45 (?)m filter and then nanof lltered : UNIT 123 (Celfa)

Membrane: DESAL DK 4040 Pressure. 1 MPa Retentate max conductivity: 12 mS/cm final conductivity: 3.5 mS/cm volume : 0.3 L Permeate max conductivity: 3.3 mS/cm final conductivity: 0.04 mS/cm volume : 5.23 L .

Time: 18 h

After adjusting the pH at 7 by adding a 1 N aqueous solution of l-deoxy-l-(methylamιno)-D-glucιtol (0.15 mL; 0.15 mmol), the retentate was loaded onto a column of Amberlite XAD 1600 polystyrene resin (40 mL ) which was eluted with H₂0 (500 mL ) . The eluate was freeze dried and then further dried (P₂0₅, 40°C, 2 kPa ) to give the title compound (42.8 g; anhydrous 40.7 g; 33 mmol). Yield 83 %. m. p. : 114°-115°C

Free ligand (0.001 M GdCl₃): 0.2% HPLC: 98 % (area %) - Chromatographic method:

Stationary phase: Eka Nobel Kromasil C4 5 μm;

250 x 4 mm column packed by Bishoff;

Temperature: 50°C; Mobile phase: gradient elution;

A = aqueous solution containing 1 g L^-1 n- hexylamine buffered at pH 6 with H₃P0₄ and 33% v/v CH₃CN

B = aqueous solution containing 1 g L" n- hexylamme buffered at pH 6 with H₃P0₄ and 55% v/v CH₃CN Gradient timetable: min % A % B

0 100 0

10 100 0

20 0 100

35 0 100 Flow rate: 1 mL mm^-1;

Detection ( UV ) : 210 nm;

Injection: 10 μL;

Sample concentration: 1 mg mL^-1;

Instrumentation : Hewlett - Packard HP 1090 M liquid chromatograph equipped with DR 5 solvent delivery system, autosampler, column thermostat and diode array detector .

K.F. : 4.80 %

MS and IR spectra were consistent with the structure. [α] -27.26° (c 2.01, H₂0)

Elemental analysis (%):

C H N Gd calcd. 46.70 6.37 5.67 12.74 found 46.70 6.14 5.77 13.00 anhydrous EXAMPLE 13 [Compound 8]

[[[4S-[4R*,8(1R*,2R*) , 12R* ]] -4-Carboxy- 5 , 11-bιs ( carbo^¬ xymethyl )-8- [ ( l-carboxy-2-methyl )butyl]-l-phenyl-12- [ (phenylmethoxy )methyl ]-2-oxa-5, 8,11-trιazatridecan-13- oate( 5- ) ]gadolmate( 2-) ] disodium salt (1:2)

10 N NaOH (4.696 mL; 46.9 mmol) was dropped into a suspension of the free ligand obtained in Example 12, Step (D) (10.35 g; 15 mmol) m H₂0 (150 mL ) stirring until a clear solution was obtained. A solution of GdCl₃-6 H₂0 (5.6 g; 15 mmol) in H₂0 (50 mL ) was slowly added maintaining the mixture at pH 7 by addition of 10 N NaOH (2.546 mL; 25.4 mmol) by means of a pH-stat apparatus. The reaction mixture was filtered through a Millipore HA 0.45 μm filter and then nano lltered : UNIT 123 (Celfa) Membrane: DESAL DK 4040 Pressure: 1 MPa Retentate max. conductivity: 6.2 mS/cm final conductivity: 2.5 mS/cm volume : 0.3 L Permeate max. conductivity: 2.2 mS/cm final conductivity: 0.04 mS/cm volume : 5.5 L

Time: 18 h

After adjusting the pH to 7 by adding 2 N NaOH (0.05 mL; 0.10 mmol) the retentate was evaporated in vacuo (2 kPa) to give the title compound (8.4 g; anhydrous 7.96 g; 8.97 mmol). Yield 60 %. m. p. : 150-152°C (synt. ) Free ligand (0.001 M GdCl₃): 0.1 %

HPLC: 97 % (area %) - Chromatographic method of Example 12, E). K.F. : 5.21 %

MS and IR spectra were consistent with the structure. [α]_D ²⁰ -28.29°(c 2.06, H₂0) Elemental analysis (%): EXAMPLE 14 [Compound 9]

[[[4S-[4R*,8(R*) ,12R*]]-4-Carboxy-5, 11 -bis (carboxymethyl )8-( 1-carboxy-pentyl )-l-phenyl-12-[ ( phenylmethoxy ) methyl ]-2-oxa-5 ,8,ll-trιazatrιdecan-13-oate(5-) ]ga- dolιnate( 2- ) ] disodium salt

A ) L-Nor leucme 1 , 1 -dιme t hy le thy 1 es t er

To a suspension of L-norleuc e (commercial product) (13.1 g; 0.1 mol) t-butyl acetate (600 L; 4.45 mol) maintained at 20°C, 70% aq. HC10₄ (10.3 mL;

0.12 mol) was added in 10 mm The reaction was maintained at r.t. for 7 h. Saturated aq. Na₂C0₃ was slowly added until pH 9 was reached and the organic phase was separated and concentrated. The oily residue was dissolved in Et₂0 (250 mL ) and extracted with IN HCl

(120 mL ) . The aqueous phase was basified to pH 10 with

IN NaOH and extracted with Et₂0 (400 mL ) . The organic layer, dried over Na₂S0₄, was concentrated to give the desired compound (14.3 g; 76 4 mmol). Yield 76 %.

TLC : R_f = 0.36

Stationary phase: silica gel plates 60 F₂^₄

Eluent: 9 : 1 CHC1₃/CH₃0H

Detection: 0.5% KMn0₄ m IN NaOH ¹³C-NMR, ¹H-NMR, MS and IP spectra were consistent with the structure.

B) N- (2-Bromoethyl)-N-[ 2- (1,1-dimethylethoxy )-2-oxo- ethyl ]-0-(phenylmethyl )-L-seπne 1 , l-dimethylethyl ester The product has been prepared according to Example 5.

C) [4S-[4R ,8(R* l2R*]]-4-[ ( 1 , 1 -Dimethylethoxy ) car- bony1 ] -8- [ 1- [ ( 1 , 1-dιmethylethoxy )carbonyl]pentyl]-5,ll- bιs[2-( 1 , 1 -dl ethylethoxy ) -2-oxoethyl ] -1-pheny1-12-

[ (phenylmethoxy )methyl ]-2-oxa-5 ,8,ll-tπazatrιdecan-13- oic acid 1 ,1-dιmethylethyl ester

„ COOtBu

"COOtBu OOOtBu

To a solution of L-nor leucme 1 , l-dimethylethyl ester (4.7 g; 25 mmol) and N-( 2-bromoethyl )-N-[ 2- ( 1 , 1- dimethylethoxy )-2-oxoethyl ]-0- (phenylmethyl )-L-seπne 1,1-dιmethylethyl ester (28 g; 60 mmol) in CH₃CN (100 mL) 2M pH 8 phosphate buffer (250 mL ) was added. The biphasic mixture was maintained under vigorous mechanical stirring for 16 h. The organic layer was separated and concentrated. The oily residue was purified by flash chromatography ( n-hexane/EtOAc 9:1 v/v) to give the desired compound (23.6 g; 24.32 mmol) Yield 97 %. TLC : R_f = 0.67

Stationary phase: silica gel plates 60 F₂c₄ Eluent: 8 : 2 n-hexane/EtOAc Detection- 254 nm; 0.5% KMn0₄ in IN NaOH ¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure.

D) [4S-[4R*,8(R*) ,12R*] ]-4-Carboxy-5,1l-bιs( carboxymethyl ) -8- ( 1-carboxypentyl ) -l-phenyl-12- [ (phenylmethoxy )methyl ]-2-oxa- 5 ,8,ll-trιazatπdecan-13-oιc acid

To a solution of the pentaester from the previous preparation (19.4 g; 19.99 mmol) in CHC1₃ (300 mL ) , maintained at 0-5°C under an inert atmosphere, (CH₃)₃SιI (40 g; 0.2 mol) was added in 2 h. The solution was allowed to rise to room temperature and left under stirring for 40 h. The solution was cooled to 0-5°C and H₂0 (150 mL ) was added. After separation the pH of the aqueous layer was adjusted to pH 3.5 with 4N NaOH. The cloudy solution was loaded onto a column of res

Amberlιte^(R) XAD 1600 (600 mL ) and eluted with H₂0 (5 L) and then with H₂0/CH₃CN (gradient elution 90:10 60:40 v/v ratios). The free ligand (9.87 g; 14.3 mmol) was obtained. Yield 72%. p : 100-103°C

HPLC: 100% (area %) - Chromatographic method: Stationary phase: Lichrospher 100 RP-8 5 μm; 250 x 4 mm column packed by Merck KGaA;

Temperature: 40°C;

Mobile phase: isocratic elution with pre ixed mobile phase: 1 g of n-octylamine is added to 300 mL of acetonitrile mixed with 700 L of water.

The solution is buffered to pH 6 with H₃P0₄;

Flow rate: 1 mL min^-1;

Detection ( UV ) : 200 nm;

Injection: 10 μL; Sample concentration: 1 mg mL ;

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (L6200 and L6000 ) , Merck KGaA -

Hitachi AS 2000 autosampler, Merck KGaA T 6300 column thermostat, Merck KGaA - Hitachi L 4250 UV detector. CE: 98.5% (area %) - Electrophoretic method:

Capillary: fused silica 0.56 m x 75 μm with bubble cell ;

Voltage: 25 kV;

Buffer: 0.05 M borate pH 9.3, EDTA 0.3 mM; Temperature: 40°C;

Stoptime: 20 min; Detection (UV): 200-210 nm;

Injection: hydrostatic (50 mbar, 5 s);

Sample concentration: 1 mg mL^-1;

Instrumentation: Hewlett Packard 3D HPCE Preconditioning timetable: t (m ) action

0 flush with H₂0

2 flush with 0.1 M NaOH

4 flush with H₂0

5 flush with buffer 9 start analysis

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure. K.F. : < 0.1% Elemental analysis (%) C H N Na

Calcd. 59.21 6.87 6.09 Found 58.85 7.26 5.84 0.11 E) [[ [4S-[4R*,8(R*) ,12R*] ] -4 -Carboxy- 5 , 11 -bis ( carboxymethyl )-8- ( 1-carboxy-pentyl )-l-phenyl-12-[( phenyl-me- thoxy) methyl ]-2-oxa--5 ,8,ll-tπazatrιdecan-13-oate(5-)]- gadolinate( 2- ) ] disodium salt

To a suspension of the free ligand from the previous preparation (4.5 g; 6.5? mmol) in H₂0 (70 L ) , maintained at 5°C, IN NaOH (13 L ) was added until a clear solution was obtained A 0.22M solution of GdCl₃ (29.7 mL; 6.54 mmol) was slowly added (1 h), maintaining the mixture at pH 7 by addition of IN NaOH. The solution was stirred for 1 h at room temperature, filtered over Millipore GSWP 0,22 , and loaded onto a column of Amberlite XAD 1600 polystyrene resm (500 L ) , which was eluted with H₂0 (1500 mL ) and then with H₂0/CH₃CN (2 L; 80:20 v/v). The title compound (5.2 g; 5.86 mmol) was obtained. Yield 90 %. mp : >250°C

HPLC: 100 % (area %) Chromatographic method of Example 3, A) .

CE : 99.5 % (area) Electrophoretic method of previous

Step D) .

Free ligand (0.001 M GdCl₃): < 0.1%

MS and IR spectra were consistent with the structure. K.F. : 6.05%

Weight loss (130°C): 6.05%

Elemental analysis (after drying at 130°C) (%):

C H N Gd Na calcd. 45.99 4.77 4.73 17.71 5.18 found 46.00 4.78 4.69 17.59 5.25 EXAMPLE 15 [Compound 10]

[[[4S-[4R*,8(R*) , 12R*]]-4-Carboxy- 5, 11-bis (carboxymethyl )-8-[l , 3-bis ( carboxy )propyl ] -1-phenyl- 12- [ (phenylmethoxy ) methyl ]-2-oxa-5 ,8 , 11-tr ιazatrιdecan-13-oate- ( 6- ) ]gadolmate ( 3- ) 3 trihydrogen compound with 1-deoxy- 1- (methylamino )-D-glucitol (1:3)

L-Glutamic acid bιs( 1 , l-dimethylethyl ) ester

(CH3)3COOC^ ^-^ ^COOC(CH3)3

NH₂ Acetic acid 1 , l-dimethylethyl ester (1742 g; 2 L; 15 mol) (commercial product), 70 % aq . perchloric acid (51 mL; 0.59 mol) and L-glutamic acid (78.8 g; 0.54 mol)

(commercial product) were stirred at 25°C over 5 days.

After addition of a solution of K₂C0 (41.46 g, 0.3 mol) in H 0 (140 mL ) to the reaction mixture, the organic phase was separated, washed with water (2 x 500 mL ) , dried (NaS0₄) and concentrated to dryness. The residue

(27.2 g) was dissolved in Et₂0 (200 mL ) and extracted with IN HCl (2 x 70 mL ) , the combined aqueous phase was washed with Et₂0 (40 L ) and collected with the aqueous phases previously obtained from the first neutralization and successive washings. Addition of 10 N NaOH up to pH

8.8 led to an emulsion which was extracted with Et₂0 (2 x 600 mL ) . The combined organic phases were washed with

H₂0 (250 L ) , dried and evaporated to give the desired compound (57.7 g; 0.22 mol). Yield 41 %.

Acidic titer (0.1 N HCl): 99,8 %

Acidic titer (0.1 N HC10₄ in CH₃COOH): 99,1 %

GC : 99.4 % (area %) - Gaschromatographic method:

Stationary phase: DB 5 (OV-73 ); Film thickness: »0.25 μm;

Column (WCOT): 30 m x 0.25 mm;

Carrier (He) flow rates: column flow rate: 0.9 mL min^-1; split flow rate: 100 mL min^-1; make up flow rate: 30 mL min^-1; septum purge flow rate: 3 mL min^-1;

Detector (FID) feeding: hydrogen pressure: 1.2 bar; air pressure: 2.8 bar;

Oven temperature timetable : initial temperature: 120°C; initial time: 2 min; rate: 10°C min^-1; final temperature: 270°C final time: 5 min,

Injector temperature: 150°C,

Detector temperature: 200°C,

Injection : 1 μL;

Sample concentration: 25 g mL^-1;

Instrumentation: Hewlett - Packard HP 5890

[α]_D ²⁰: +20.83° (c 2.0; MeOH)

¹³C-NMR, ¹H-NMR and MS spectra were consistent with the structure.

Elemental analysis (%):

C H N Calcd. 60.20 9.72 5.40 Found 60.33 9.70 5.35 B) N-(2-Bromoethyl)-N-[2-(l,l-dιmethylethoxy)-2-oxo- ethyl]-0-(phenylmethyl )-L-serme

The product has been prepared according to Example 5. C) [43-[4R*,8(R*) , 12R*]] -4- [(1,1-Dimethylethoxy) car- bony1] -8- [4- (1 ,1-dimethylethoxy )-l-[ ( 1 , 1-dimethyletho- xy)carbonyl]-4-oxobutyl]-5,ll-bιs[2-(l , 1-dimethylethoxy )-2-oxoeth l ]-l-phenyl-l2- [ (phenylmethoxy ) methyl] -2- oxa-5 ,8 ,ll-triazatrιdecan-13-oic-acid 1 , 1-dιmethylethyl ester

Phosphate buffer 2 M, pH 8 (1700 m ) was added to a solution of N-( 2-bromoethyl )-N-[2-( 1 ,1-dιmethylethoxy )- 2-oxoethyl]-0-(phenylmethyl)-L-serιne 1 , 1-dimethylethyl ester (171.2 g; 0.36 mol) and L-glutamic acid bιs(l,l- dimethylethyl) ester (44.1 g; 0.17 mol) in CH₃CN (1450 mL ) . After 15 h of vigorous stirring the two phases were separated and further 2 M pH 8 phosphate buffer (900 mL ) was added to the organic phase. After 27 h the organic phase was separated and evaporated under reduced pressure (2 kPa ) . The residue was dissolved in CH C1₂

(1700 L ) and the resulting solution was washed with water (900 mL ) , dried (Na₂S0₄) and concentrated. The crude was dissolved in n-hexane (1000 mL ) to give a precipitate (Ph₃P=0) which was filtered off. The solution was evaporated to dryness (2 kPa ) and the residue was purified by flash chromatography (Stationary phase: silica gel 230 - 400 mesh ASTM; Eluent: n-hexane and from 9 : 1 to 5.7 : 1 n-hexane/ EtOAc ) to give the desired product (154 g, 0.15 mol) Yield 87 %. Acidic titer (0.1 N HCl): 100 0 % HPLC: 96.2 % (area %) - Chromatographic method: Stationary phase: -Merck KGaA Lichrosorb RP-Select B 5 μm; 250 x 4 mm column packed by Merck KGaA; Temperature: 35°C; Mobile phase: gradient elution, A = 0.017 M H₃P0₄ m water

B = CH₃CN Gradient timetable: min % A % B

0 70 30 40 20 80 50 20 80

Flow rate: 1 L min^-1; Detection (UV): 210 nm;

Injection: 10 μL;

Sample concentration: 1 mg L —1 ;

Instrumentation : Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck KGaA - Hitachi L 3000 diode array detector. TLC: Rf = 0.36 Silica gel plates 60 F₂^₄ Eluent: 4 : 1 n-hexane/EtOAc

Detection: UV (254 nm) and 1% KMn0₄ in 1 M NaOH

Weight loss (60°C): < 0.10 %

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure. [α]_D ²⁰: -31.26° (c 5.03, CHC1₃) Elemental analysis (%):

C H N calcd. 65.68 8.79 4.03 found 65.78 9.11 4.10 D) [4S-[4R*,8(R* ) , 12R* ] ]-4-Carboxy- 5 , 11-bιs ( carboxymethyl )-8-[l , 3-bιs( carboxy )propyl] -1-phenyl-12- [ (phenylmethoxy ) methyl ]-2-oxa-5 , 8 , ll-trιazatπdecan-13-oιc acid

CF₃COOH (40.6 mL; 60.5 g; 0.53 mol) was added to a solution of the hexaester from the previous preparation

(151.7 g; 0.14 mol) in CH₂C1₂ (530 mL ) maintaining the temperature at 0°-5°C. The resulting solution was evaporated (2 kPa ) , the residue was dissolved in CF₃COOH (264 L; 393 g; 3.45 mol) and the solution stirred at room temperature for 15 h. After evaporation (2 kPa) the residue was again dissolved in CF₃COOH (100 mL; 150 g; 1.3 mol) in order to complete the reaction. The resulting mixture was stirred at room temperature for 14 h and then evaporated under reduced pressure (2 kPa). The crude (243.7 g) was dissolved in 1 : 5 CH₃CN/H₂0 (550 L ) and loaded onto a column of Amberlite XAD 1600 polystyrene resin (1.9 L) which was eluted with CH₃CN/H₂0 to give the desired compound (89.2 g; anhydrous 87.2 g; 0.124 mol). Yield 85 %. m.p. : 109 - 110'C

Acidic titer (0.1 N NaOH): 100.9 %

Complexometric titer (0.1N ZnS0₄ ) : 98.8 %

HPLC: 97.4 % (area %) Chromatographic method of previous

Step C) . Weight loss (90"C)_#: 2.29 %

¹³C-NMR, ---H-NMR, MS and IR spectra were consistent with the structure.

[α]_D ²⁰: - 17.07° (c 2.06, 0.4 M NaOH)

Elemental analysis (%): C H N calcd. 56.16 6.14 5.95 found 56.61 6.29 6.07 anhydrous E) £[[4S-[4R*,8(R*) , 12R*]]-4-Carboxy-5, ll-bis( carboxymethyl) -8- [1 ,3-bis( carboxy )propyl]-l-pheny1-12-[ (phe- nylmethoxy ) ethyl] -2-oxa-5 ,8,11-triazatridecan-13-oate-

( 6- ) ]gadolinate( 3- ) ] trihydrogen compound with 1-deoxy- l-(methylamιno)-D-glucιtol (1:3)

A I M aqueous solution of 1-deoxy-l- (methylamino )-

D-glucitol (154 mL; 0.154 mol) was dropped into a suspension of the product from the previous preparation (28.2 g; 0.040 mol) in H₂0 (150 mL ) , stirring until a clear solution was obtained. A solution of GdCl₃ ^• 6 H₂0

(14.9 g; 0.040 mol) in H₂0 (40 mL ) was slowly added maintaining the mixture at pH 7 by addition of a 1 M aq solution of l-deoxy-l-(methylammo)-D-glucιtol (91.5 mL ; 0.092 mol) by means of a pH-stat apparatus. The reaction mixture was filtered through a Millipore HA 0.45 μ filter and then nanof lltered :

UNIT 123 (Celfa)

Membrane: DESAL DK 4040 Pressure: 1 MPa

Retentate max conductivity: 11.5 mS/cm final conductivity: 5.1 mS/cm volume: 0.3 L Permeate max conductivity: 3.0 mS/cm final conductivity: 0.13 mS/cm volume: 2.8 L

Time: 15 h The retentate ( pH 7.1) was freeze dried at first and then further dried (P₂0₅, 2 kPa ) to give the title compound (52.4 g; anhydrous 51.8 g; 0.036 mol).

Yield 90%. m. p. : 116 - 118°C Free ligand (0.001 M GdCl₃): < 0.10 %

HPLC: 97 % (area %) - Chromatographic method: Stationary phase: Spheri -10 RP-2 10 μm;

250 x 4,6 mm column packed by Applied Biosystem; Temperature: 40°C; Mobile phase: isocratic elution: A/B = 80 : 20 ;

A = 1 g of n-octylamme is added to 1000 L of water. The solution is buffered to pH = 6 with H₃P0₄ ;

B = CH₃CN Flow rate: 1.0 mL min -1. Detection ( UV ) : 210 nm; Injection: 10 μL;

Sample concentration- 1 mg mL^{' ■}1

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (two Lachrom L 7100 pumps), Merck

KGaA - Hitachi Lachrom L 7200 autosampler, Merck KGaA

- Hitachi Lachrom L 7300 column thermostat, Merck

KGaA - Hitachi Lachrom L 7400 UV detector.

K.F. : 1.21 %

MS and IR spectra were consistent with the structure.

[α]_D ²⁰: - 24.29° (c 2.01, H₂0)

Elemental analysis (%):

C H N Gd calcd. 44.87 6.34 5.81 10 88 found 44.62 6.44 5.80 10 79 anhydrous EXAMPLE 16 [Compound 11]

[[ [N,N' -[ [ ( Carboxymethyl )ιmιno ]dι -2,1 -ethanediyl ]bιs[N- ( carboxymethyl )-L-phenylalanιnate ] ] ( 5- ) ]gadolιnate( 2- ) ] dihydrogen compound with 1-deo y-l -( methylamino ) -D-glu- c i to l ( 1 . 2

A) N-(2-bromoethyl)-N-[2-(l , 1-dimethylethoxy )-2-oxo- ethyl]L-phenylalanme 1 , l-dimethylethyl ester

The product is prepared according to Example 3.

B) Glycme 1 , l-dimethylethyl ester

The product is prepared according to Example 1. C) N,N'-[[ [2- (1,1-Dimethylethoxy )-2-oxoethyl]ιmιno]dι- 2 ,lethanedιyl]bιs[N-[2-(l , 1-dιmethylethoxy )-2-oxoethyl ]- L-phenylalanme 1 , l-dimethylethyl ester]

A two phase mixture of N-( 2-bromoethyl )-N-[2- ( 1 , 1- di ethylethoxy )-2-oxoethyl ]-L-phenylalanιne 1 ,1-dimethylethyl ester (116.4 g; 0.221 mol) and glycme 1,1- dimethylethyl ester (13.6 g; 0.104 mol) in MeCN (1000 m ) and 2 M pH 8 phosphate buffer (600 mL ) was stirred for 20 h. The upper layer was separated and replaced with fresh 2 M pH 8 phosphate buffer (400 L ) . The reaction mixture was stirred for additional 14 h. The upper layer was separated and the solvent evaporated. The residue was dissolved in n-hexane (500 L ) and the solution washed with H₂0 (400 L ) . After drying (Na₂S0₄), the solvent was evaporated under reduced pressure (2 kPa ) and the residue (107 g) purified by flash chromatography:

Sample: Solid dispersion m silica gel 35-70 mesh

Stationary phase : 1200 g Silica gel 230-400 mesh Stationary phase conditioning: n-hexane

Eluent: n-hexane/EtOAc gradient

(v/v) Volume (L)

100/0 1.5

95/5 1 92.5/7.5 2

90/10 2

87.5/12.5 2

85/15 6.5

After further drying (P 0₅; 0.13 kPa; 50°C) the desired product was obtained (71.9 g; 0.084 mol) . Yield

81 %.

Acidic titer (0.1 N HCl) : 98.2 %

TLC : Rf 0.3

Stationary phase: Silica gel plates 60 F₂^₄ Eluent: -4:1 (v/v) n-Hexane/EtOAc

Detection: 1 % KMn0₄ in 1 N NaOH

HPLC : 98.5 % (area %) Chromatographic method:

Stationary phase: Lichrosorb RP-Select B 5 (?)m;

250 x 4 mm column packed by Merck KGaA; Temperature: 45°C;

Mobile phase: gradient elution;

A = 0.017 M H₃P0₄ in water

B = CH₃CN

Gradient timetable: mm % A % B 0 82 18

30 15 85 45 15 85

Flow rate: 1 mL min^-1;

Detection ( UV ) : 210 nm;

Injection: 10 μL; Sample concentration: 1 mg mL^-1;

Instrumentation : Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck

KGaA - Hitachi L 3000 diode array detector. Weight loss (70°C high vacuum) : 0.66 %

¹³C-NMR, ¹H-NMR, MS and IR spectra were consistent with the structure .

[α]²⁰ (c 5.12; CHC1₃ > ^λ(nm> 589 578 546 436 405 365 r ι ²⁰ 1 *J ; - 19 . 56 ° - 20 . 55 ° - 23 . 52 ° - 42 . 18 ° - 51 . 58 ° - 71 . 27 °

Elemental analysis (%):

C H N

Calcd. 67.50 8.85 4.92 Found 67.27 8.82 4.98 D) N,N'-[[(Carboxγmethyl)imιno]dι-2,l-ethanediyl]- bιs[N-( carboxymethyl )-L-phenylalanιne]

Trifluoroacetic acid (96.3 g; 0.845 mol) was dripped, over 30 mm, into a solution of the product obtained at the previous step (33.3 g; 0.039 mol) in CH₂C1₂ (20 mL) stirred at -15 ÷ -10°C. The cooling bath was removed and the reaction stirred at room temperature for 64 h. The solvent was removed by evaporation under reduced pressure (2 kPa ) and the residue taken up with fresh trif luoroacetic acid (96.3 g; 0.845 mol). After additional 26 h stirring, the reaction mixture was evaporated under reduced pressure (2 kPa ) obtaining a brown oil that was dissolved in CH₂C1₂ (100 mL ) and again evaporated in vacuo. This operation was repeated twice. Following the same procedure the residue was treated with Et₂0 (200 L ) and then with CH₃CN (50 mL ) obtaining a brownish amorphous solid that was dissolved in 1/1 (v/v) CH₃CN/H₂0 (50 mL ) acidified with 37% HCl

(10 L) and diluted to 200 mL with H₂0. The solution was loaded onto a column of Amberlite^ XAD 1600 (1 L) and the product eluted with a H₂0/CH₃CN gradient:

Conditioning: 90/10 (v/v) H₂0/CH₃CN Elution: H₂0/CH₃CN gradient

(v/v) Volume (L)

90/10 3.5

80/20 2.5

75/25 1.5 70/30 1.5

65/35 1.5

60/40 1

40/60 1

20/80 1.5 The product elutes with 75/25 (v/v) H₂0/CH₃CN.

Fractions containing the pure ligand were collected and evaporated in vacuo (2 kPa ) to give a solid residue that was dried overnight (2 kPa; P 0₅; 35°C). This solid was suspended in CH₃CN (100 mL ) and stirred for several hours, filtered and dried (2 kPa; P₂0₅; 35°C) to afford the desired product (19.72 g; 0.0345 mol). Yield 88 %. m . p . : 111-115 ' C

Acidic titer (0.1 N NaOH) : 96.2 %; equivalent point pH

6.54

Complexometric titer (0.1 N ZnS0₄ ) 96 %

HPLC : 99.5 % (area %)

Weight loss (70°C high vacuum) : 1.58 %

¹³C-NMR, ¹H-NMR, MS and IR spectra are consistent with the structure.

[α]²⁰ (c 2.5; 0.4 N NaOH; ^λ(™"⁾ 589 578 546 436 405 365

20

[°-]A + 10.68° + 11.60° + 13.32° + 26.79° + 36.35° + 58.47' Elemental analysis (%)

C H N

Calcd. 58.63 6.15 7.33 Found 58.61 6.36 7.97

E) [ [ [N,N'-[ [ ( Carboxymethyl )ιmmo]dι-2,1-ethanediyl] - bιs[Ncarboxymethyl )-L-phenylalanιnate] ] ( 5-) ]gadolma- te(2-)] dihydrogen compound with 1-deoxy-l- (methyl- ammo )-D-glucιtol (1:2)

To a solution obtained suspending the product obtained at the previous step (14 34 g; 0.025 mol) in H₂0 and neutralizing (pH 7.0) by addition of 1 N aq meglumine (83 mL; 0.083 mol), was added, over 15 min, a solution of GdCl₃ 6 H₂0 (8.92 g; 0.024 mol) m H₂0 (100 mL) maintaining the pH at 7 with 1 N aq. meglumine (37 mL; 0.037 mol) by means of a pH-stat apparatus. The reaction mixture was nanoflltered : Apparatus: Celfa Unit C-123-P

Membrane: Desal DK 4040

Pressure: 1 MPa

Time: 17 h Conditions: volume starting final conductivity conductivity retentate 0.35 L 11 mS/cm 2.9 mS/cm permeate 5.5 L 2.8 mS/cm 0.03 mS/cm and then freeze-dried . After further drying (2 kPa;

P₂Or^; 40°C) the title compound was obtained (24.5 g;

0.0219 mol) . Yield 87 %. m.p. : 135-139^*C

HPLC : 99.2 % (area %) - Chromatographic method: Stationary phase: Spheri -10 RP-2 10 mm;

250 x 4,6 mm column packed by Applied Biosystem;

Temperature: 45°C;

Mobile phase: isocratic elution with premixed mobile phase: 1 g of n-octylamine is added to 240 mL of acetonitrile mixed with 760 mL of water. The solution is buffered to pH 6 with H₃P0₄;

Flow rate: 1.0 mL min^-1;

Detection (UV): 210 nm;

Injection: 10 ml; Sample concentration: 1-10 mg mL^-1;

Instrumentation: Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck

KGaA - Hitachi L 3000 diode array detector. K.F. : 1.86 %

MS anc . R spectra were consistent with the structure. Elemental analysis (%)

C H Gd N Calcd. 45.11 5.95 14.06 6.26 Found 44.80 5.97 14.15 6.26 anhydrous

EXAMPLE 17 [Compound 12]

[ [ [S-(R*,R* )]-N,N-Bis[ 2- [(carboxymethyl )( l-carboxy-2- phenylethyl) amino] ethyl ]-L-phenylalaninate( -) ]gadoli- nate(2-)] dihydrogen compound with l-deoxy-l-(methyl- amino )-D-glucitol (1:2)

A) N- (2-Bromoethyl)-N- [2- (1,1-dimethylethoxy )-2-oxo- ethyl]-L-phenylalanine 1 , l-dimethylethyl ester

The product is prepared according to Example 3. B) [S-(R*,R*)]-N,N-Bis[2-[[2-(l,l-dimethylethoxy)-2- oxoethyl] [2-( 1 , 1-dimethylethoxy )-2-oxo-l- (phenylme- thyl ) ethy1] amino ] ethyl ]-L-phenylalanine 1 , l-dimethylethyl ester

A two-phase mixture of N- ( 2-Bromoethyl )-N-[2-( 1 , 1- di ethylethoxy )-2-oxoethyl ]-L-phenylalanιne 1, 1-dime- thylethyl ester (68 g; 0.15 mol) and L-phenylalanine 1 , l-dimethylethyl ester (Example 3, Step A) (13.3 g; 0.06 mol) in MeCN (800 mL ) and 2 M pH 8 phosphate buffer (500 L ) was stirred for 17 h. The upper layer was separated and further 2 M pH 8 phosphate buffer (200 mL ) was added. After 24 h, the upper layer was separated and the solution was evaporated. The residue was dissolved in n-hexane (500 mL ) and the solution was washed with H₂0 (300 mL ) . After drying (Na₇S0₄), the solution was evaporated and the residue was purified by flash chromatography (Stationary phase- silica gel 230-400 mesh Merck KGaA art 9385 (1 kg) Eluent: 10 : 1 n- hexane/EtOAc ) to give the desired product (48 g ; 0.051 mol) . Yield 85 % TLC : Rf 0.50 Stationary phase : Silica gel plates 60 ?254 (Merck KGaA code 5715)

Eluent: n-hexane/EtOAc 4 : 1 Detection: 1 % KMn0₄ in IN NaOH HPLC : 96.9 % (area %) - Chromatographic method: Stationary phase: Lichrosorb RP-Select B 5 μm; 250 x 4 mm; column packed by Merck KGaA; Temperature: 45°C;

Mobile phase: gradient elution;

A = 0.01 M KH₂P0₄ and 0.017 M H₃P0₄ m water

B = CH₃CN Gradient timetable: min % A % B

0 95 5

30 20 80

55 20 80

Flow rate: 1 mL min^-1; Detection ( UV ) : 210 nm;

Injection: 10 μL;

Sample concentration: 1 mg mL^-1;

Instrumentation : Merck KGaA - Hitachi high pressure gradient pump system (two Lachrom L 7100 pumps), Merck KGaA - Hitachi Lachrom L 7200 autosampler, Merck KGaA

- Hitachi Lachrom L 7300 column thermostat, Merck KGaA

- Hitachi Lachrom L 7400 UV detector.

¹H-NMR,¹³C-NMR, MS and IR spectra are consistent with the structure. [ [αα]]²^^0υ c 5.14, CHCI

λ(nm) 589 578 546 436 405 365 tα]_Λ - 39.25° - 41.53° - 47.85° - 85.20° - 104.83° no transmi_ttance

Elemental analysis (%): C H N

Calcd. 69.96 8.65 4.45

Found 69.83 8.31 4.21 C) [ S-(R*,R*) ]-N,N-Bιs[ 2- [(carboxymethyl ) ( l-carboxy-2- phenylethyl )amino] ethyl]-L-phenylalanme

Trimethylsilyl iodide (commercial product) (80 g; 0.40 mol) was dripped, in 40 mm, into a solution of the compound obtained from the previous preparation (37.8 g; 0.040 mol) in CHC1₃ (200 mL ) maintained under stirring at 5 10°C. After 19 h the mixture was dropped in H₂0/ιce (600 g). The amorphous precipitate was dissolved in MeOH (200 L ) and 2N NaOH (100 mL ) was added to the solution up to pH 7. The solution was concentrated to remove methanol and dropped into 1 N HCl (250 mL ) containing NaHS0₃ (1 g). The precipitate (containing iodide ions) was dissolved in CH₃CN (80 L ) and the solution was diluted with H₂0 (300 L ) and few drops of 37% HCl to maintain the solution clear. The solution was loaded on to a column of Amberlite XAD-1600 (1 L), eluted with 4 : 1 CH₃CN/0.1 N HCl ust before and after the loading to prevent the precipitation of the product on to the res . The column was washed with 4 : 1 CH₃CN/H₂0 (2 L), then the product was eluted with 1 ^• 1 CH₃CN/H₂0 (3 L). The eluate was evaporated at 40°C and 1 3 kPa and the residue was treated with H₂0 (50 mL ) to afford a crystalline solid. After filtration and drying (P₂Or, 50°C, 2 kPa) the desired product was obtained (19.6 g; 0.0295 mol). Yield 74 % mp : 116°C synt . ; 142°C dec Acidic titer (0.1 N NaOH) : 97.9 %; equivalent point pH

7.4

HPLC : 97.3 % (area %) - Chromatographic method of previous step B ) .

K.F. : 0.87

¹H-NMR, ¹³C- MR, MS and IR spectra were consistent with the structure.

[α]²⁰ (c 2.53; 4 N NaOH)

λ(nm) 589 578 546 436 405 365 20 f^αh + 14.57° + 15.33' 17.58' +^' 31.49° + 38.69° + 52.28°

Elemental analysis (%):

C H N Calcd. 63.34 6.23 6.33 Found 63.28 6.02 6.39 anhydrous D) [[[S-(R*,R*)]-N,N-Biε[2-[ ( carboxymethyl )( 1-carboxy- 2-phenylethyl ) amino ]-ethy1 ] -L-phenylalaninate( 5- ) ]gado- linate(2-)] dihydrogen compound with 1-deoxy-l- (methylamino)-D-glucitol (1:2)

A solution of GdCl₃ ^• 6 H₂0 (8.18 g; 0.022 mol) in H₂0 (50 mL ) was dripped over 3 h into a solution of the compound obtained in the previous step (14.6 g; 0.022 mol) neutralised ( pH 7.0) by addition of 1 N meglumine (70 L; 0.07 mol). The reaction mixture was maintained at pH 7 with 1 N meglumine (37.6 L; 0.038 mol) by means of a pH-stat apparatus. The complexation was monitored by HPLC.

Chromatographic method: Stationary phase: Lichrospher 100 RP-8 5 (?)m;

250 x 4 mm column packed by Merck KGaA;

Temperature: 40°C;

Mobile phase: isocratic elution with pre ixed mobile phase: 1 g of n-nonylamme is added to 400 mL of μacetonitrile mixed with 600 L of water. The solution is buffered to pH 6 with H₃P0₄; Flow rate: 1 mL mm^-1;

Detection ( UV ) : 210 nm; Injection: 10 μL; Sample concentration: 1 mg mL^-1;

Instrumentation: Merck KGaA - Hitachi high pressure gradient pump system (two Lachrom L 7100 pumps), Merck KGaA - Hitachi Lachrom L 7200 autosampler, Merck KGaA - Hitachi Lachrom L 7300 column thermostat, Merck KGaA - Hitachi Lachrom L 7400 UV detector.

The solution was filtered through Millipore HA 0.45 m and loaded on to Amberlite^' XAD 1600 (1000 mL ) , the resin was washed with H₂0, 1:9 MeOH/H₂0, 1:4 MeOH/H₂0, 2:3 MeOH/H₂0, 4:1 MeOH/H₂0 and the product was eluted with MeOH. After evaporation of methanol, the pH of the solution was corrected to 6.8 with 1 N meglumine (0.4 m ) and the solution was concentrated to dryness at 40°C and 1.3 kPa . After drying (P₂0₅, 50°C, 270 Pa) the title compound (23.9 g; anhydrous 23.0 g; 0.195 mol) was obtained as a white solid. Yield 89 %. mp : 56°C (143°C synt . ) HPLC : 99.7 % (area %)

K.F. ^• 3.63 %

The MS spectrum is consistent with the structure

Elemental analysis (%):

C H Gd N

Calcd. 48.70 6.01 13.01 5.80

Found 49.46 6.24 13.30 5.88 anhydrous

EXAMPLE 18 [Compound 13]

[[[[S-(R*,R*)]-α,α'-[[ ( Carboxymethyl ) ιmmo]bιs[ 2 , 1- ethanediylf ( carboxymethyl )ιmmo] ] ]bιs [cyclohexane- propanoate] ] ( 5- ) ]gadolιnate( 2- ) ] dihydrogen compound with l-deoxy-l-(methylammo )-D-glucιtol (1:2)

A) N,N'-[[[2-(l ,1 -Dime thy lethoxy )-2-oxoethyl ] ιmmo]dι- 2 , l-ethanedιyl]bιs[N-[2-( 1 , 1 -dime thy lethoxy )-2-oxo- ethyl]-L-phenylalanme 1 , l-dimethylethyl ester]

The product s prepared according to Example 16, Step (C).

B) [S-(R*,R*)]-α,α'-[[[2-(l,l-Dιmethγlethoxγ)-2-oxo- ethyl]ιmmo]bιs[2 , 1-ethanedιyl [ [ 2-( 1 , 1-dimethylethoxy ) - 2-oxoethyl]ιmmo] ] ]bιs-[cyclohexanepropanoιc acid 1,1- dimethylethyl ester]

To a solution of N ,N ' - [ [ [ 2- ( 1 , 1-Dιmethylethoxy )-2- oxoethyl]imιno]di-2 , 1-ethanedιyl ]bιs[N-[ 2- ( 1 , 1-dιme- thylethoxy )-2-oxoethyl]-L-phenylalanme 1 , 1-dimethylethyl ester] (35.9 g; 0.042 mol) in MeOH (300 L ) , wet 5% Rh on carbon (commercial product) (8 g) was added and the resulting suspension was hydrogenated , in a Parr bomb (mod. 4561, 600 mL vessel) (theoretical hydrogen 5.64 L; 0.252 mol), at 4xl0⁶ Pa (40 bar) and at 60-65°C for 6 h. The mixture was cooled, the catalyst was removed by filtration through buchner funnel and the filtrate was evaporated under reduced pressure. The residue was dissolved m absolute EtOH (500 L ) , concentrated to dryness, dissolved in n-hexane (500 mL ) and again concentrated under reduced pressure. The residue (36 g) was purified by flash chromatography. Sample: Solid dispersion in silica gel 35-70 mesh Stationary phase : 1200 g Silica gel 230-400 mesh Stationary phase conditioning: n-hexane Eluent: n-hexane/EtOAc gradient

(v/v) Volume (L)

100/0 1

97.5/2.5 1

95/5 2

92.5/7.5 2

90/10 2

87.5/12.5 2

85/15 3 to give, after further drying (P₂0₅; 0.13 kPa; 50°C), the desired product (30.5 g; 0.035 mol). Yield 83 %.

Acidic titer (0.1 N HCl) : 98.3 %; equivalent point pH

4.49 TLC : Rf 0.52

Stationary phase: Silica gel plates 60 F₂₅₄

Eluent: 4:1 (v/v) n-hexane/EtOAc

Detection: 1 % KMn0₄ in 1 N NaOH

HPLC : 98.5 % (area %) Chromatographic method:

Stationary phase: Lichrosorb RP-Select B 5 (?)m;

250 x 4 mm column packed by Merck KGaA;

Temperature: 45°C;

Mobile phase: gradient elution; A = 0.017 M H₃P0₄ in water

B = CH₃CN

Gradient timetable: min % A % B

0 82 18

30 15 85 45 15 85

Flow rate: 1 L mm^-1;

Detection ( UV ) : 210 nm;

Injection: 10 μL;

Sample concentration: 1 mg mL^-1; Instrumentation : Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck

KGaA - Hitachi L 3000 diode array detector.

Weight loss (70°C high vacuum) : 1.59 % ¹H-NMR, ¹ C-NMR, MS and IR spectra were consistent with the structure. [ α ] 1 220U ( c 5 . 43 , CHC1₃ >

λ(nm) r [α i] 2_Λ0

Elemental analysis (%):

C H N Calcd. 66.56 10.12 4 85 Found 66.82 10.28 4 88

C) [S-(R*_/R*)]-α,α'-[[ (Carboxyme hyl ) ιmιno]bιs [ 2 , 1- ethanedιyl-[ (carboxymethyl ) imino] ] ]bιs [ cyclohexane- propanoic acid]

Trif luoroacetic acid (commercial product) (81.4 g; 0.714 mol) was dripped, over 30 mm, into a solution of the pentaester obtained in the previous step (29 g; 0.0335 mol) m CH₂C1₂ (20 L ) stirred at -15 ÷ -10^*C The cooling bath was removed and the reaction stirred at room temperature for 90 h The solvent was removed by evaporation under reduced pressure (2 kPa) and the residue taken up with fresh tri f luoroacetic acid (81 4 g; 0.714 mol). After additional 50 h stirring, the reaction mixture was evaporated under reduced pressure (2 kPa) to give a brown oil that was dissolved in CH₂C1₂ (100 L) and again evaporated in ^acuo This operation was repeated twice. Following the same procedure the residue was treated with Et₂0 (2 x 100 mL ) and with CH₃CN (50 mL), obtaining a brownish amorphous solid that was dissolved in 1/1 (v/v) CH₃CN/H₂0 (50 mL ) , acidified with 37% HCl (10 mL ) and diluted to 200 L with H₂0 The solution was loaded onto a column of Amberlιte^R XAD 1600

(1 L) and the product eluted with a H₂0/CH₃CN gradient:

Conditioning: 90/10 (v/v) H₂0/CH₃CN Elution: H₂0/CH₃CN gradient

(v/v) Volume (L)

90/10 4.5

80/20 1.5

70/30 2 60/40 3.5

50/50 1.5

20/80 1.5

The product elutes with 60/40 (v/v) H₂0/CH₃CN

Fractions containing the pure ligand were collected and concentrated in vacuo (2 kPa) The azeotropic removal of CH₃CN led to the separation of a precipitate that was filtered, washed with H₂0 and dried overnight

(2 kPa; P₂0₅; 40°C) to afford the desired product (14.71 g; 0.025 mol) . Yield 75%. m.p. : 116-120°C

Acidic titer (0.1 N NaOH) : 96.3 % ; equivalent point pH

7.24

Complexometric titer (0.1 N ZnS0₄ ) : 96.5 %

HPLC : 99 % (area %) - Chromatographic method of previous step B)

K.F. : 2.29 %

!H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

[α]²⁰ (c 2.5; 0.4N NaOH) Λ λ(nm) 589 578 546 436 405 365 r _T 20 l^a + 4 . 87 ° + 5 . 15 ° + 5 . 67 ° + 9. 19 ° + 12 . 58 ° + 19 . 73 ° Elemental analysis (%): C H N Cl- F Calcd. 57.42 8.09 7.17 Found 56.16 8.39 7.00 < 0.1 < 0.1 Corresp. to 57.47 8.32 7.16 anhydrous D) [[[[S- (R*,R*)3-α,α'-[[ (Carboxymethyl )ιmιno]bιs[ 2,1- ethanedιyl[ (carboxymethyl )ιmino] ] ]bis[cyclohexanepropano ate] ] ( 5- ) ]gadolinate ( 2- ) ] dihydrogen compound with 1- deoxy-l-(methylamιno)-D-glucιtol (1:2)

To a solution obtained suspending the product obtained in the previous step (12.6 g; 0.0215 mol) in H₂0 and neutralising (pH 7.0) by addition of 1 N aq meglumine (75 mL; 0.075 mol), was added, over 15 min, a solution of GdCl₃ 6 H₂0 (7.8 g; 0.021 mol) in H₂0 (100 mL ) maintaining the pH at 7 with 1 N aq meglumine (30 mL; 0.03 mol) by means of a pH-stat apparatus. The reaction mixture was nanofiltered Apparatus: Celfa Unit C 123 P Membrane: Desal DK 4040 Pressure: 1 MPa Time: 15 h Conditions:

and then freeze-dried . After further drying kPa; P₂0₅; 40°C) the title compound was obtained (22.5 g;

0.0199 mol) . Yield 92 %. m.p.: 145-150°C

HPLC : 99.9 % (area %) - Chromatographic method: Stationary phase: Spherι-10 RP-2 10 μm;

250 x 4,6 mm column packed by Applied Bioεystem;

Temperature: 45°C;

Mobile phase: isocratic elution with pre ixed mobile phase: 1 g of n-octylamme is added to 350 mL of acetonitrile mixed with 650 mL of water. The solution is buffered to pH 6 with cone. H₃P0₄;

Flow rate: 1.0 L mm^-1;

Fluorimeter Detection: Ex 275 nm , Em 315 n ;

Injection: 10 μL; Sample concentration: 5-10 mg mL^-1;

Instrumentation: Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck

KGaA - Hitachi F 1080 Fluorescence Detector K.F. : 1.14 %

MS and IR spectra were consistent with the structure.

Elemental analysis (%):

C H Gd N

Calcd 44.63 6.96 13.91 6.20 Found 44.35 6.91 14.00 6.21 anhydrous

EXAMPLE 19 [Compound 14]

[[[[[aS-[aR*(a^,R*),(lR*)]]-a,a'-[[(l-Carboxy-2-cyclo- hexylethyl )ιmmo]bιs[2 , 1-ethanedιyl [ (carboxymethyl )- imino] ] ]bιs[cyclohexanepropanoate] ] ( 5- ) ]gadol ate( 2- ) ] dihydrogen compound with 1-deoxy-l- ( methylamino )-D-glu- citol (1:2)

A) [S-(R*,R*)]~N,N-Bis[2-[[2-(l , 1 -dimethylethoxy ) -2- oxoethyl] [2-( 1 , 1 -dimethylethoxy )-2-oxo-l- (phenylme- thyl )ethyl]ammo]ethyl]-L-phenylalanme 1, l-dimethylethyl ester

The product is prepared according to Example 17, Step

(B).

B) [α-[R*(α'R*), ( IR* ) ] ] -α , α ' - [ [ 1- ( 1 , 1-Dιmethyletho- xy )carbonyl-2-cyclohexylethyl]ιmιno]bιs [2,1-ethanediyl-

[ ( 1 , 1-dimethylethoxy )-2-oxoethyl] )ιmιno]bιs[cyclo- hexanepropanoic acid 1 , 1-dιmethy lethy 1 ester]

To a solution of [S-(R ,R ) ] -N , N-bis [ 2- [ [ 2- ( 1 , 1- di e thy lethoxy )-2-oxoethyl ][2-(l ,l-dιmethylethoxy)-2- oxo-l-(phenylmethyl )ethyl]ammo]ethyl ]-L-phenylalanme 1, l-dimethylethyl ester (67.4 g; 0.07 mol) in MeOH (500 mL ) , 5 % Rh on carbon (commercial product) (13.4 g) was added and the resulting suspension was hydrogenated

(theoretical hydrogen 14.1 L; 0.63 mol) at 40 bar (4

MPa) and at 60-65°C for 6 h. The reaction was monitored by HPLC (Chromatographic method of Example 17, B)). The mixture was cooled and the catalyst was removed by filtration on Buchner funnel. The filtrate was concentrated under reduced pressure and the residue was purified by f lash-chromatography (Stationary phase: S llica gel 230-400 mesh Merck KGaA art 9385 (600 g) Eluent: 100:1 30:1 n-hexane/EtOAc gradient) to give the desired product (51.9 g; 0.054 mol). Yield 77 % TLC : Rf 0.80

Stationary phase: Silica gel plates 60 ?254 (Merck KGaA code 5715) Eluent: 4 : 1 n-hexane/EtOAc

Detection: 1 % KMn0₄ in IN NaOH or Pancaldi spray (CeS0₄ ^• 4 H₂0 1 g; ( NH₄ ) ₆Mo_?0₂₄ ^• 4 H₂0 21 g; 98 % H₂S0₄ 31 mL; H₂0 470 mL ) and heating to 200°C ¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

[α]²⁰ (c 5.00; CHC1 A λ(nm) 589 578 546 436 405 365 r ι ²⁰

^[ot] - 42.92° - 45.88° - 51.29° - 90.17° - 111.23° - 152.08'

Elemental analysis (%):

C H N

Calcd. 68.64 10.37 4.37

Found 68.83 10.74 4.26

C) [αS-[αR*(α'R*) , ( IR* ) ] ]-α , α ' - [ [ ( l-Carboxy-2-cy- clohexylethyl ) imino]bis[2 ,l-ethanedιyl[ (carboxymethyl )- imino] ] ]bis[cyclohexanepropanoιc acid]

Tπmethylsilyl iodide (commercial product) (106 g; 0.53 mol) was dripped, in 40 min, into a solution of the compound obtained in the previous step (51 g; 0.053 mol) in CHC1₃ (250 m ) maintained under stirring at 5 10°C. After 88 h the mixture was dropped in H₂0/ιce (300 g). The amorphous precipitate was dissolved by the addition of 2 N NaOH until pH 8 was reached, the solution was evaporated to remove CHC1₃ and acidified with 18% HCl (to pH 2) to obtain an orange precipitate, that was filtered. The solid was dissolved in 30% MeOH (2 L) and 10 N NaOH (to pH 10), Na₂S0₃ (500 mg ) was added and the solution was acidified with 18 % HCl (to pH 4,8) The solution was loaded on to a column of Amberlιte(^R) XAD 1600 resm ( 050 mm; h 920 mm; 1800 mL ) and washed with 3:6:1 MeOH/H₂0/ 37% HCl (1 L), 3.7 MeOH/H₂0; 1:1 MeOH/H₂0, 7:3 MeOH/H₂0, 9:1 MeOH/H₂0, MeOH. The product was then eluted with 9 1 MeOH/25% aq. NH₃. The eluate was concentrated to remove MeOH and NH₃ and the residual solution (300 mL ) was acidified with 37% HCl The precipitate (gel) was filtered, washed five times with H₂0 (to remove Cl^") and dried (P₂0₅, 50°C, 2 kPa ) . The desired product was obtained (27.5 g, 0 0403 mol). Yield 76 % mp : 161*C (synt.) 169°C (dec.)

Acidic titer (0.1 N NaOH)' : 94 %; equivalent point pH 7.9

Complexometric titer (0.1 N ZnS0₄ ) : 93.5 %

K.F. : 3.69 %

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure .

[α]²⁰ (c 2.50; 4 N NaOH)

A λ(nm) 589 578 546 436 405 365

20

W7 + 37.02° + 38.74° + 44.17° + 73.64° + 88.90° + 118.53°

Elemental analysis (%):

C H N Cl^'

Calcd. 61.65 8.72 6.16

Found 62.11 8.76 6.23 < 0.10 anhydrous D) [[[[[αS-[αR*(α*R*) , ( IR* ) ] ]-α , α ' - [ [ ( l-Carboxy-2- cyclohexylethyl ) ιmιno]bιs[2 , 1-ethanedιyl [ (carboxymethyl ) l mo] ] ]bιs[cyclohexanepropanoate] ] ( 5- ) ]gadolι- nate (2-)] dihydrogen compound with 1-deoxy-l- ( methylamino ) -D-glucitol (1:2).

A solution of GdCl₃ ^■ 6 H₂0 (commercial product) (13 g; 0.035 mol) in H₂0 (50 mL ) was dripped over 3 h into a solution of the product obtained at the previous step (23.8 g; 0.035 mol) in H₂0 (100 mL ) neutralised ( pH 7.0) by addition of 1 N meglumine (95.6 L; 0.096 mol) The reaction mixture was maintained at pH 7 with 1 N meglumine (72.3 mL; 0.072 mol) by means of a pH-stat apparatus. The complexation was monitored by titration (12). The mixture was filtered through Celite and

Millipore HA 0.45 μm and loaded on to a column of Amberlite XAD 1600 resin (-050 mm; h 230 mm; 450 mL ) . The resin was washed with H₂0 and the product was eluted with 1:4 MeOH/H₂0. After evaporation of methanol, the pH of the solution was corrected to 6.8 with 1 N meglumine (0.2 L ) and the solution was concentrated to dryness at 40^*C and 1.3 kPa . After drying (P₂°5_' 50°C, 270 Pa) the title compound was obtained (33.2 g; anhydrous 31.5 g ; 0.025 mol) . Yield 73 %. mp : 168°C (125°C synt. ) K.F. : 5.2 %

The MS spectrum was consistent with the structure. Elemental analysis(%):

C H Gd N Calcd. 47.98 7.40 12.82 5 . 71 Found 48.28 7.31 12.69 5 . 6 3 anhydrous EXAMPLE 20 [Compound 15]

[[[[[αS-[αR*(α'R*),(lR*}]] —α , α ' - '-[ [ ( 1 -Carboxy-3- phenylpropyl )imino]biε[2,i-ethanedιyl [ ( carboxymethyl ) imino] ]]bis[benzenebutanoate]](5-)]gadolinate(2-)] dihydrogen compound with 1-deoxy-l- ( ethylamino )-D- gluci tol ( 1 : 2 )

A) ( S )-α-Aminobenzenebutanoιc acid 1 , l-dimethylethyl ester (C.A.S. [83079-77-0])

The compound has been prepared according to- Haslanger, M. F.; Sybertz , E. J.; Neuεtadt, B. R . ; Smith, E. M.; Nechuta, T. L.; Berger , J. J. Med . Chem. 1989, 32(4), 737-739.

B) ( S)-α-[[2-(l, 1-Dιraethylethoxy )-2-oxoethyl](2-hy- droxyethyl )ammo]benzenebutanoιc acid 1 , l-dimethylethyl ester

A solution of (S)-α-amιnobenzenebutanoιc acid 1,1- di ethylethyl ester (42.4 g; 0.18 mol), 2-(2-bromo- ethoxy )tetrahydropyran, prepared according to: J. Org. Chem. 1986, 51, 752-755, (39.7 g; 0.19 mol) and diisopropylethylamine (commercial product) (34 L; 0.2 mol) in CH₃CN was refluxed for 21 h. Diisopropylethylamine (37.5 mL; 0.22 mol) and tert-butyl bromoacetate (commercial product) (39 g; 0.2 mol) were added and the reaction mixture refluxed for further 2.5 h. The solution was evaporated under reduced pressure (2 kPa ) to give a residue that was dissolved in n-hexane (0.5 L) and washed with H₂0 (4 x 0.5 L). The organic phase was separated, treated with Carbopuron^ ^{R )} 4 N, filtered, dried over Na₂S0₄ and evaporated under reduced pressure. The residue (90 g) was dissolved in cool (5°C) MeOH (0.4 L) and cool (5°C) 2N HCl (0.2 L) was added. After 4 h at r.t., cool (5°C) 2N NaOH (0.25 L) and subsequently n-hexane (0.5 L) were added. The upper layer was separated and evaporated under reduced pressure to give a residue that was dissolved m n- hexane (0.5 L). The solution was washed with H₂0 (2 x 0.25 L), dried over Na₂S0₄ and evaporated under reduced pressure. The residue (66 g) was purified by flash chromatography :

Sample: 1:1.5 (w/w) solid dispersion in silica gel 35-

70 mesh Stationary phase : 1 kg silica gel 230-400 mesh

Stationary phase conditioning: n-hexane

Eluent: n-hexane/EtOAc gradient

(v/v) Volume (L)

100/0 1 95/5 1

90/10 2

85/15 2

80/20 7

After further drying (P₂0₅; 0.13 kPa; 50°C) the desired product was obtained (38.8 g; 0.098 mol). Yield

55 %.

Acidic titer (0.1 N HC10₄ ) : 100.7 %

TLC : Rf 0.18

Stationary phase: Silica gel plates 60 F₂^₄ Eluent: 3:1 (v/v) n-hexane/EtOAc

Detection: 1 % KMn0₄ in 1 N NaOH

HPLC : 99 % (area %) - Chromatographic method:

Stationary phase: Lichroεorb RP-Select B 5 μm;

250 x 4 mm column packed by Merck KGaA; Temperature: 45°C;

Mobile phase: gradient elution; A = 0.017 M H₃P0₄ in water

B = CH₃CN

Gradient timetable: min % A % B

0 82 18 30 15 85 45 15 85

Flow rate: 1 L min^-1;

Detection (UV): 210 nm;

Injection: 10 μL;

Sample concentration: 1 mg L -^"1.

Instrumentation : Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck

KGaA - Hitachi L 3000 diode array detector.

Weight loss (70°C, high vacuum) : 1.10 %

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

[α]²⁰ (c 5.14; CHC1 ) Λ 3

589 578 546 436 405 365

-22.61° -23.68° -27.24° -48.28° -59.19° -80.34

Elemental analysis (%):

C H N Calcd. 67.15 8.96 3.56 Found 67.65 9.31 3.75

C) (S)-α-[(2-Bromoethyl)[ 2- (1,1-dιmethylethoxy )-2- oxoethyl]amino]benzenebutanoιc acid 1 , l-dimethylethyl ester

N-Bromosuccmimide (commercial product) (24.1 g;

0.13 mol) was added in a single portion to a solution of the product from the previous preparation (37 g, 0.094 mol) and triphenylphosphine (commercial product) (34.1 g; 0.13 mol) in CH₂C1₂ (250 L ) cooled at -60°C by means of EtOH-dry ice bath. After 5 mm the bath was removed and the reaction was allowed to stand at r.t. for 2 h

After addition of ice (200 g), 5% aqueous NaHCO (100 mL ) and CH C1₂ (100 mL ) , the organic phase was separated, washed with 5% aqueous NaHCO-, (200 mL ) , H_?0

(200 mL ) , dried over a₂S0₄ and evaporated under reduced pressure (2 kPa ) . The residue was suspended in n-hexane

(300 mL ) , the solid precipitate (triphenylphosphine oxide) was filtered off, washing with cool n-hexane (500 mL ) , and the filtrate concentrated under reduced pressure (2 kPa ) to 300 L Carbonpuron ' ^{R )} 4N was added and the solution, cooled at 5°C, filtered The filtrate was evaporated under reduced pressure (2 kPa ) to give the desired product (43 1 g) Argentometric titer (0.1 N AgN0₃ ) 94 3% TLC : Rf 0.50

Stationary phase: Silica gel plates 60 F₂^₄ Eluent: 85:15 (v/v) n-hexane/EtOAc

Detection: 1% KMn0₄ 1 N NaOH D) [αS-[αR*(α'R*) , ( IR* ) ] ]-α , α ' - [ [ 1- [ ( 1 , 1-Dιmethyl- ethoxy )carbonyl]-3-phenylpropyl ]ιmιno]bιs[2 ,1-ethane- diyl [1-(1 ,1-dimethylethoxy )-2-oxoetnyl]ιmιno]bιs[ben- zenebutanoic acid 1 , 1-dιmethy lethyl ester]

A two phase mixture of ( S )-α- [ ( 2-bromoethyl ) [2- ( 1 , 1-dimethylethoxy )-2-oxoethy1] amino] benzene butanoic acid 1,1-dιmethylethyl ester (43.1 g; 0.089 mol), (S)-α- ammobenzene butanoic acid 1 , l-dimethylethyl ester (9.9 g; 0.042 mol) in MeCN (400 mL ) and 2 M pH 8 phosphate buffer (250 mL ) was stirred for 8 h. The aqueous layer was separated and replaced with fresh 2 M pH 8 phosphate buffer (200 mL ) . The same operation was repeated after 27 h and 52 h. The reaction mixture was stirred for additional 20 h (72 h total). The upper layer was separated and the solvent evaporated. The residue was dissolved in Et₂0 (500 L ) and the solution washed with H₂0 (2 x 250 mL). After drying (Na₂S0₄), the solvent was evaporated under reduced pressure (2 kPa ) . The residue was purified by flash chromatography:

Sample: 1:1.5 solid dispersion in silica gel 35-70 mesh Stationary phase : 500 g silica gel 230-400 mesh Stationary phase conditioning: n-hexane Eluent: n-hexane/EtOAc gradient (v/v) Volume (L) 100/0 1

98/2 1 96/4 1

92/8 1 90/10 2.5

85/15 6.5

After further drying (10 Pa; 40°C) the desired product was obtained (39.5 g; 0.04 mol). Yield 95 %.

Acidic titer (0.1 N HCl) : 95.3 % ; equivalent point pH

3.6

TLC : Rf 0.36

Stationary phase: Silica gel plates 60 F₂₅₄

Eluent: 85:15 (v/v) n-hexane/EtOAc

Detection: 1% KMn0₄ in 1 N NaOH

HPLC : 99 % (area %) Chromatographic method of previous step B) .

Weight loss (70°C, high vacuum) : 0.61 %

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure.

[α] ° (c 5.04, CHC1 ) λ(nm) 589 578 546 436 405 365 r i 2 2.0 -48.01° -50.33° -57.69° -103.15° -126.78° -174.94°

Elemental analysis (%):

C H N Calcd. 70.63 8.89 4.26

Found 71.35 8.97 4.32

E) [αS-[αR*(α'R*) , ( IR* ) ] ]-α , α ' - [ [ ( l-Carboxy-3-phe- nylpropyl ) imino]bis [2 , 1-ethanediyl [ (carboxymethyl )- imino] ] ]bis [benzenebutanoic acid]

Trifluoroacetic acid (14.8 g; 0.13 mol) was dropped, over 15 mm, into a solution of the pentaester from the previous preparation (39.1 g; 0.039 mol) in

CH₂C1₂ (50 mL) stirred at 0°C. The solution was evaporated under reduced pressure (2 kPa ) , the residue dissolved in trif luoroacetic acid (74 g; 0.65 mol) and the reaction mixture stirred at r t. for 18 h. The solvent was removed by evaporation under reduced pressure (2 kPa) and the residue taken up with fresh trif luoroacetic acid (74 g; 0.65 mol) After additional

72 h stirring, the solvent was evaporated affording an oil that was dissolved m CH₂C1₂ (100 ) and again evaporated m vacuo. This operation was repeated twice.

Following the same procedure the residue was treated with Et₂0 (2 x 100 L ) and then with CH₃CN (100 mL ) to give an amorphous solid that was dissolved in 3/2 (v/v)

CH₃CN/H₂0 (200 mL), acidified with 37% HCl (10 mL ) and diluted to 400 mL with H₂0. The resulting solution was loaded onto a column of Amberlιte(^R) XAD 1600 (1 L) and the product eluted with a H₂0/CH₃CN gradient:

Conditioning: 90/10 (v/v) H₂0/CH₃CN

Elution: H₂0/CH₃CN gradient

(v/v) Volume (L)

90/10 2.5 80/20 2

70/30 1

60/40 2

55/45 4

50/50 1 40/60 2

The product elutes with 60/40 (v/v) H₂0/CH₃CN. The fractions containing the pure product were combined and evaporated under reduced pressure . On mixed fractions this procedure was repeated a second time in order to get a further amount of pure product. After further drying (2 kPa; P2°5'' 40°C) the desired compound

(21.1 g; 0.029 mol) was obtained. Yield 74 %. mp : 115-120'C

Acidic titer (0.1 N NaOH) : 98%; equivalent point pH 7.0

Complexometric titer (0.1 N ZnS0₄ ) : 96 % HPLC : 97 % (area %) Chromatographic method of previous step B) .

K.F. : 0.86 %

¹H-NMR, ¹³C-NMR, MS and IR spectra were consistent with the structure. [α]²⁰ (c 2.51; 0.4 N NaOH)

λ(nm) 589 578 546 436 405 365 <-^ai λ + 6.64° + 6.80° + 7.00° +11.18° +14.28° +23.07°

Elemental analysis (%): C H N

Calcd. 64.67 6.71 5.95

Found 64.01 6.79 6.13 anhydrous

F) C[[[[αS-[αR*(α'R*) , ( IR* ) ] ]-α , α ' - [ [ ( l-Carboxy-3- phenylpropyl )imino]bis[2,l-ethanedιyl[ ( carboxyme- thyl)imino]] ]bis [benzenebutanoate ] ](5-)]gadolinate(2-)] dihydrogen compound with 1-deoxy-l- ( methylamino )-D- glucitol (1:2)

To a solution obtained suspending the free ligand from the previous preparation (18.4 g; 0.025 mol) in H₂0 (50 mL) and neutralizing ( pH 7.0) by addition of 1 N aq. meglumine (80 L; 0.08 mol), was added dropwise a solution of GdCl₃-6 H₂0 (8.92 g; 0.024 mol) in H₂0 (20 L ) maintaining the pH at 5 with 1 N aq. meglumine (40 mL; 0.04 mol) by means of a pH-stat apparatus. The reaction mixture was neutralized to pH 7 with 1 N aqueous meglumine, then nanof lltered :

Apparatus: Celfa Unit C-123-P

Membrane: Desal DK 4040

Pressure: 1 MPa

Time: 15 h Conditions:

Volume Starting Final conductivity conductivity

Retentate 0.3 L 11.3 mS/cm 2.7 mS/cm

Permeate 5.8 L 2.3 mS/cm 0.03 mS/cm and finally freeze-dried . After further drying (2 kPa;

P₂0₅; 40°C) the title compound (21.4 g; 0.017 mol) was obtained. Yield 68 %. mp : 133-138°C

Free metal (0.001 M EDTA) : > 0.005 % HPLC : 99.2 % (area %) - Chromatographic method:

Stationary phase: Spheri -10 RP-2 10 μm;250 x 4,6 mm column packed by Applied Biosystem;

Temperature: 40°C;

Mobile phase: isocratic elution with pre ixed mobile phase: 1 g of n-octylamine is added to 300 mL of acetonitrile mixed with 700 mL of water. The solution is buffered to pH 6 with cone. H₃P0₄;

Flow rate: 1.0 mL min^-1;

Detection (UV): 210 nm; Injection: 10 μL;

Sample concentration: 1 mg mL^-1; Instrumentation : Merck KGaA - Hitachi L 6200 low pressure gradient pump, Merck KGaA - Hitachi AS 2000 autosampler, Merck KGaA T6300 column thermostat, Merck

KGaA - Hitachi L 3000 diode array detector. K.F. : 3.57 %

MS and IR spectra were consistent with the structure.

Elemental analysis (%):

C H Gd N

Calcd. 49.95 6.29 12.58 5.60 Found 50.27 6.23 12.17 5.81 anhydrous

Claims

1. Compounds of formula (I), either in their racemic or enantiomeric forms:

wherein :

R is H or a linear or branched, saturated or unsaturated ^Cι^-C20 alkyl chain, which is interrupted or not by one or more 0, N, S atoms or by one or more -CO-, -CH(OH)-, -CH(NH₂)-, -CONH-, -NHC0-, -SO-, -S0₂-, -S0₂NH-, which is substituted or not with one or more halogen atoms or -COOH groups or their ester or amide derivatives and which is interrupted or not or substituted or not by one or more cyclic R₃ residues which can be the same or different and isolated or fused, with the proviso that, if some of said residues are fused, the maximum number of rings forming the corresponding polycyclic unit is three, in which:

R₃ is a 5- or 6-membered carbocyclic or heterocyclic , saturated, unsaturated or aromatic cyclic unit, substituted or not with one or more groups X, which can be the same or different, in which:

X is OH, halogen, NH₂ , NHL, N(L)₂, -0-L, -S-L, -CO-L, where L, the same or different from each other, is C^-Cς linear or branched alkyl, substituted or not with one or more hydroxy, alkoxy or carboxylic groups, or X is a COOH group or its ester or amide derivative, or a -S0₃H group or its amide derivative, and R^ , R₂ have the same meanings as R, independently from each other, except H, with the proviso that: when R^ and R₂ are both C₆H₅-CH₂~0-CH₂- , R is different from either H or C₆H₅-CH₂-0-CH₂-; as well as the complexes of the compounds of formula (I) with metal ions having atomic number from 20 to 31, 39, from 42 to 44, 49 and from 57 to S3 and the salts thereof with physiologically acceptable organic bases selected from primary, secondary or tertiary amines, or basic amino acids, or with inorganic bases the cations of which are sodium, potassium, magnesium, calcium or mixtures thereof.

2. Compounds as claimed m claim 1, wherein the meanings of R, R-. and R₂ are selected from the following ones :

CH.

CH, ".

-CM. -CH, -CH,

^?CH, CH, CH,

3. Compounds as claimed in claim 1, wherein the co plexed metal ion is selected from Fe( ⁺), Fe(³⁺), _Cu(2+) _f cr(³⁺), Gd( ⁺), Eu(³⁺), Dy(³⁺), La< ⁺>, Yfa( ⁺) and Mn<²⁺) .

4. Compounds as claimed in claim 1, of formula (II):

(ID where :

R₄ = H, or a linear or branched ^cι^_C₁₀ alkyl, optionally interrupted by one or more -CONH-, -NHC0-, -CO- groups and/or N, 0, S atoms, optionally interrupted or substituted with 1 to 3 saturated rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with -OH, -SH, halogen, -COOH, - H₂, -N(R")₂, -C0N(R")₂, -S0₃H, C₁ -C₄ alkoxy groups;

R₅ = independently a linear or branched ^cι^-cιo alkyl, optionally interrupted by one or more -CONH-, -NHCO-, -CO- groups and/or N, 0, S atoms and interrupted or substituted with 1 to 3 saturated rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with -OH, -SH, halogen, -COOH, -NH₂, -N(R*')₂, -C0N(R")₂, -S0₃H, C^^ alkoxy groups;

R" = independently H or ^{c _c}5 linear or branched alkyl, optionally substituted with from 1 to 5 -OH groups .

5. Compounds as claimed in claim 1, of formula (III)

(Ill) where

= H, or a linear or branched ^cι^-cιo alkyl, optionally interrupted by one or more -CONH-,

-NHC0-, -CO- groups and/or N, S atoms and optionally substituted w th one or more -OH, -NH₂ ,

-COOH groups;

R. = independently a linear or branched C₂-C-,_Q alkyl, optionally interrupted by one or more -CONH-,

-NHCO- , -CO- groups and/or N, S atoms and optionally substituted with one or more -OH, -NH-,,

-COOH groups.

Compounds as claimed in claim 1, of formula (IV):

(IV) where

R₈ = H, or a linear or branched C^-C^n alkyl, optionally interrupted by one or more -CONH-,

-NHCO-, -CO- groups and/or N, S atoms, optionally interrupted or substituted with 1 to 3 isolated or fused saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with one or more -OH, -COOH, -NH₂ , -N(R")₂, ^-Cg alkyl, C₁-C₅ alkoxy, Cg-C₂₀ arylalkoxy groups; Rg = independently a linear or branched C₁-C_& alkyl, optionally interrupted by one or more -CONH-, -NHCO-, -CO- groups and/or N, S atoms, which is interrupted or substituted with 2 to 3 fused saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with one or more -OH, -COOH, -NH₂, -N(R" )₂, C₁-Cg alkyl, ^cl^-c6 ^alkoχY' ^C6^-C20 a^rY^lalkoxy groups;

R" = independently H or C₁-C₅ linear or branched alkyl, optionally substituted with 1 to 5 -OH groups. 7. Compounds as claimed in claim 1, of formula (V):

( V ) where :

R₁₀ = a linear or branched C;L^_CIO ^alJζY¹/ optionally interrupted by one or more -CONH-, -NHCO- , -CO- groups and/or N, S atoms, interrupted or substituted with 1 to 3 saturated, unsaturated or aromatic rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with one or more -OH, -COOH, -NH-, , -N⁽R") / ^cι^-c6 ^alkv1' ^ci"^c6 ^alkoχY groups;

^Rll ⁼ independently ^a linear or branched C₂-C_1Q alkyl, optionally interrupted by one or more N, S atoms.

8. Compounds as claimed in claim 1, of formula (VI):

( VI ) where :

R₁₂ = a linear or branched C₂-C₁₀ alkyl, optionally interrupted by one or more -CONH-, -NHCO- , -C0- groups and/or N, S atoms, optionally substituted with one or more -COOH, - H₂ groups, optionally interrupted or substituted with 1 to 3 saturated, unsaturated or aromatic, isolated or fused rings, that are optionally interrupted by one or more N, 0, S and that are optionally substituted with one or more -OH, -COOH, -NH₂, -N(R")₂, C_±-C₆ alkyl, C^-Cg alkoxy groups. 9. Compounds as claimed in claim 1, of formula (VII):

( VII ) where :

R₁₃ = H, linear or branched C-^-Cg alkyl, substituted or interrupted with 1 aromatic ring, that is optionally interrupted by one or more N, 0, S;

R₁₄ = independently linear or branched C₁-C₈ alkyl, substituted or interrupted with one aromatic ring, that is optionally interrupted by one or more N, 0, S.

10. Compounds as claimed in claim 1, of formula (VIII):

( VI I I ) where :

^R15 ⁼ independently H, halogen;

R₁₆ = H, OH, N(R")₂, COOR" , -C0N(R")₂, -S0₃H, -S0₂NHR" ,

^Cl^-C6 ^allcY¹' ^cl^-c6 ^al^koχY'^' R₁₇ = independently ^Cι^-C6 alkyl, substituted with -COOH or -C0N(R")₂ or from 1 to 3 -OH groups;

A = direct bond (i.e. no intervening atom), -O- , C=0 = integer 1-6; n = integer 0-2;

R" = independently H or ^Cι^-C5 linear or branched alkyl, optionally substituted with 1 to 5 -OH groups with the proviso that, when R₁₆ = H, at least one of the substituents R₁₅ is different from hydrogen. 11. Compounds as claimed in claim 1 to 10, selected from the following group: - [4S-[4R*,8(R*) ,12R*]]-4-Carboxy-8-[l-carboxy-2-(4- hydroxyphenyl ) ethyl ] -5 ,11-bιs ( carboxymethyl ) -1-phenyl- 12- [ (phenylmethoxy )methyl]-2-oxa- 5 , 8, 11-trιazatridecan- 13-oic acid;

[4S-[4R*,8(1R*,2R*) , 12R*] ] -4-Carboxy-5 , 11-bis ( car- boxymethyl) -8-[ ( 1-carboxy-2-methyl ) butyl ]-l-phenyl-12- [ (phenylmethoxy )methyl ]-2-oxa- 5 ,8 , ll-trιazatπdecan-13- oic acid;

N,N'-[ (Carboxymethylιmmo)dι-2 , l-ethanediyl]bis[N- carboxymethyl-L-isoleucine ] ; - [1S-[1R*(1R*,2R*) , 2R* ]-N , N-Bis [ 2- [( carboxymethyl )- ( l-carboxy-2-methylbutyl ) amino] ethyl ]-L-ιsoleucine;

N,N'-[ (Carboxymethylimmo)dι-2 , l-ethanediyl]bis[N- carboxymethyl-L-tryptophan] ;

[S-(R*,R*)]-N,N'-[[(l-Carboxy-2-methylbutyl)ιmιno]- di-2,lethanedιyl]bis[N-carboxymethyl-L-tryptophan] ;

[1S-[1R*(1R*,2R*) ,2R*]]-N,N-Bιs[2-[ ( carboxymethyl )- ( 1-carboxy-2-methyl-butyl ) amino ] ethy1 ] -L-tyrosine;

[4S-[4R*,8(R*) ,12R*] ]-4-Carboxy-5 , 11-bis ( carboxymethyl) -8-( 1-carboxypentyl )-l-phenyl-12-[ (phenylmethoxy )- methyl]-2-oxa-5 ,8 ,ll-triazatrιdecan-13-oιc acid;

[4S-[4R*,8(R*) ,12R*] ]-4-Carboxy-5, 11-bis ( carboxyme- thyl )-8-[l, 3-bis(carboxy)ρropyl]-l-phenyl-12-[ (phenylmethoxy )methyl ]-2-oxa-5, 8,11-triazatr^"idecan-13-oic acid;

N,N' -[ [ (Carboxymethyl )imino]di-2, l-ethanediyl]bis- [N-( carboxymethyl )-L-phenylalanine] ; - [S-(R*,R*)]-N,N-Bis[ 2- [(carboxymethyl) ( l-carboxy-2- phenylethyl)amino] ethyl]-L-phenylalanine;

[S-( *,R*)]-α,α' -[[(Carboxymethyl ) imino] is [2, 1- ethanediyl-[ (carboxymethyl ) imino3 ] ]bis[cyclohexanepropa- noic acid]; - [αS-[αR*(α^,R*),(lR*)]]-α,α*-[[(l-Carboxy-2-cyclohe- xylethyl)imino]bis[ 2, l-ethanediyl[ (carboxymethyl ) imino]- ] ]bis[cyclohexanepropanoic acid] .

12. A paramagnetic chelate as claimed in claim 1 to 10, selected from the following group: - [[[4S-[4R*,8(R*) , 12R* ] ]-4-Carboxy-8- [ l-carboxy-2- ( 4-hydroxyphenyl)ethyl]-5, 11-bis (carboxymethyl )-1-phe- nyl-12-[ (phenylmethoxy )methyl]-2-oxa-5 ,8 , 11-triazatride- can-13-oate( 5- ) ]gadolinate( 2-) ] dihydrogen compound with 1-deoxy-l-methylamino-D-glucitol (1:2); - [[[4S-[4R*,8(lR*,2R*),12R*]^]-4-Carboxy-5,ll-bis-

( carboxymethyl )-8- [ ( l-carboxy-2-methyl )butyl ]-l-phenyl- 12- [ (phenylmethoxy ) ethyl]-2-oxa-5, 8 , 11-triazatridecan- 13-oate( 5- ) ]gadolinate( 2-) ] dihydrogen compound with 1- deoxy-1- (methylamino )-D-glucitol (1:2); - [[[-N,N'-[ (Carboxymethylimino)di-2,l-ethanediyl]- bis[N-carboxymethyl-L-isoleucinate] ] ( 5- ) ]gadolinate( 2- ) ] disodium salt;

[[[1S-[1R*(1R*,2R*) ,2R*]]-N,N-Biε[ 2- [(carboxymethyl ) ( 1-carboxy-2-methylbutyl) amino]ethyl ]-L-isoleucina- te-( 5- ) ]gadolinate(2- ) ] disodium salt;

[ [ [-N,N'-[ (Carboxymethylimino)di-2,l-ethanediyl]- bis[N-carboxymethyl-L-tryptophanate] ] ( 5- ) ]gadolina- te(2-)] disodium salt;

[ [ [ [S- ( R* , R* ) ]-N,N ' - [ [ ( l-Carboxy-2-methylbutyl ) imino]di-2 , 1-ethanediy1]bis [N-carboxymethyl-L-tryptophana- te] ]-( 5-) ]gadolinate(2- ) ] disodium salt;

[[[lS-[lR*(lR*,2R*),2R*]]-N,N-Bis[2-[( carboxymethyl ) ( l-carboxy-2-methylbutyl )amino]ethyl]-L-tyrosinate- ( 5- ) ]gadolinate( 2- ) ] disodium salt;

[ [[4S-[4R*,8(R*), 12R*]]-4-Carboxy-5, 11-bis (carboxy- methyl )8-( 1-carboxy-pentyl )-l-phenyl-12-[ (phenylmethoxy )methyl]-2-oxa-5 ,8 ,ll-triazatridecan-13-oate( 5- ) ]gado- linate(2-)] disodium salt;

[[[4S-[4R*,8(R*) , 12R*]]-4-Carboxy-5, 11-bis (carboxymethyl)-8-[1 , 3-bis ( carboxy )propyl]-1-pheny1-12- [ (phenyl- methoxy) ethyl]-2-oxa-5 , 8 , ll-triazatridecan-13-oate-

( 6- ) ]gadolinate( 3- ) ] trihydrogen compound with 1-deoxy- l-( methylamino )-D-glucitol (1:3);

[ [[N,N'-[[ (Carboxymethyl ) imino]di-2, 1-ethanediyl] - bis [N-( carboxymethyl )-L-phenylalaninate] ] ( 5-) ]gadolina- te(2-)] dihydrogen compound with 1-deoxy-l- (methylamino )-D-glucitol (1:2);

[ [[S-(R*,R* )]-N,N-Bis [2- [(carboxymethyl )(l-carboxy- 2-phenylethyl )amino]ethyl]-L-phenylalaninate( 5- ) ]gadoli- nate(2-)] dihydrogen compound with 1-deoxy-l- (methyl- amino )-D-glucitol (1:2);

[[[[S-(R*,R*)]-α,α*-[[(Carboxymethyl)imino]bis[2,l- ethanediyl[ (carboxymethyl ) imino] ] ]bis[cyclohexanepropa- noate] ] ( 5-) ]gadolinate( 2- ) ] dihydrogen compound with 1- deoxy-l-(methylamino)-D-glucitol (1:2); - [[[[[aS-[aR*(a^,R*),(lR*)33-a,a^*-[[(l-Carboxy-2-cy- clohexylethyl)imino]bis[2,l-ethanediyl[ (carboxymethyl)- imino] ] ]bιs[cyclohexanepropanoate] ] ( 5- ) ]gadolιnate( 2- ) ] dihydrogen compound with l-deoxy-l-(methylammo)-D-glu- citol (1:2).

13. Compounds as claimed claims 1 to 10 and 12, further characterized in that the relaxivity values (r^ r₂) in human serum reconstructed with Seronorm™ Human, at a concentration comprised from 0 to 1 mM, at 20 MHz and 39°C, is higher or the same as 15 s^-1mM^_1.

14. A contrast diagnostic pharmaceutical composition for Magnetic Resonance Imaging comprising at least one of the complex chelates as claimed in claims 1 to 10 and 12 or a physiologically acceptable salt thereof.

15. A pharmaceutical composition as claimed in claim 14, for imaging of human or animal body organs and/or tissues, by use of Nuclear Magnetic Resonance.

16. The use of the complex chelates of the compounds as claimed in claims 1 to 12, or of the salts thereof, for the preparation of diagnostic formulation for M.R.I. , for obtaining images of human or animal body organs and/or tissues by use of Nuclear magnetic Resonance.