DK170946B1 - Nitrogen-containing cyclic ligands, metal complexes formed by these ligands, diagnostic compositions containing these complexes, and methods for preparing the ligands - Google Patents

Abstract

Ligands and complexes of the title and the applications of these complexes in magnetic resonance imaging, in X-ray radiology and as in-vivo chemical shift agents.

Description

in DK 170946 B1

The present invention relates to novel nitrogen-containing cyclic ligands and metal complexes formed by these ligands, diagnostic agents containing these complexes as magnetic resonance imaging agents, as X-ray contrast agents, and as reagents providing chemical shifts in vivo.

The invention also relates to a method for preparing the ligands.

EP No. 124,766 discloses contrast agents for enteral administration comprising a paramagnetic compound in combination with a buffer and an osmotically active substance.

From the document it appears that complexes of the formula Ic

X-CH2 xCH2 "X

'N-CH.-CH, -N

in 2 2 »5H 2 C, H, N-CH.-CH.-M / 2 2 \ x-ch 2 ch 2-x can be used as the paramagnetic compound. In the document, the use of 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) is proposed as complexing acid to prepare the complex salts of formula Ic, cf. page 9, line 5 of the specification, and as an example of suitable paramagnetic metal ions is suggested. on page 5, the line 10 gadolinium (III) ion.

DE 3,401,052 relates to a diagnostic agent comprising, as an essential component, at least one complex salt derived from a ligand and one or more metals having atomic numbers 21 to 29, 42, 44 or 57 to 83. Imaging is particularly highlighted by gadolinium (III), terbium (III), dys-prosium (III), holmium (III) and erbium (III) ions. Regarding the structural formula of the complex salt, it is proposed in DE 25 no. the following formula Ic DK 170946 B1 2

X-CH2 / CH2-X

'N-CH - CH - N

i 2 2 \ CH2 CH, (CH-) (CH ~) * 2 «/ 2 v N-CHO-CH, -N / 2 2 \ x-ch2 ch2-x

It is noted that Example 11 (page 40) relates to the gadolinium (III) complex of 1,4,7,10-tetraazacyclododecantetraacetic acid (DOTA).

WO 86/02352 discloses an NMR contrast agent comprising a gadolinium chelate having a compound selected from DOTRA, DOTA and NOTA.

The nitrogenous ligands according to the invention differ from the ligands known from the above-mentioned writings in that at least one of the bridging chains, i.e. R1 # which connects two nitrogen atoms carries a substituent, whereas no such substituent is present at a corresponding site in the known ligands.

As can be seen from the following Comparative Example 13, this structural difference is reflected in the fact that the metal complexes between ligand and metal ion referred to herein have o-inferior properties compared to the known metal complexes in the form of lower toxicity and lower dissociation constant.

In the case of NMR imaging, the stability of the metal or ligand complex or chelate is essential. One of the purposes of using a metal chelating ligand is precisely to avoid the release of the paramagnetic metal, which in the free state is extremely toxic.

Thus, the magnitude of the dissociation constant of the ligand-metal complex reflects the resulting toxicity. More specifically, the higher the dissociation constant, the higher the toxicity.

The invention relates to a ligand of the formula:

R R

I "i *

H00C - CH CH - C00H

/ - \ (I) R3 "i \ / N- R -2 I 2

H00C-CH

R 5 where 5 R 1 represents a radical of the formula: “(CH 2) m - CH - CH -

I I

1 * 6 R7

R 9 is selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 3 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl and a group of the formula: R 1

CH-C00H CH-C00H

Γ N - R1 - N

r J- \ R, - f "*, -" '“Γ / - ^ /

2 - R - N

2 I

CH-C00H I

R5.

Rn is selected from the group consisting of Group A and the groups of formula: DK - 170946 B1 4 - (CH2) t - Y - A - Y - (CH2) t - A is selected from the group consisting of C1-C8 alkylene, C the hydroxyalkylene and Cj-Cg polyhydroxyalkylene, Y are selected from - C - O - and - O and t = 1 to 4,

II

o R 7 is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 3 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl, m = 0 or 1, R 2, r 3, r 4 are the same or different and represent a radical of the formula: - (CH) - CH -

I I

Rg R $ 15 Rg and R8 are the same or different and are selected from hydrogen, C1-C4 alkyl, C1-C4 hydroxyalkyl and C1-C4 polyhydroxyalkyl, p is 1 or 2, n is 0, 1 or 2. , and

R 1 is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, 20 C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl, and Z is selected from the group consisting of oxygen and a group of the formula: \ N - R10 25 / R10 is selected from the group consisting of hydrogen, C 1 -C 14 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 -poly hydroxyalkyl, a group of the form DK 170946 B1 5 -CH - COOH, wherein R 5 is as defined previously, and a% group of the formula: P «. R- i 5 i 3

CH-COH CH-COOH

/ - 'Jr \ \ /'

- R. - N -— R _ - N

12. 2 I

CH-COOH

* 5 · R 12 is selected from the group consisting of C 1 -C 8 alkylene, C 1 -C 8 hydroxyalkylene and C 1 -C 8 polyhydroxyalkylene, as well as salts thereof.

The ligands of formula I can be prepared by reacting a compound of formula: X - CH - COOH (II)% wherein R 5 is as defined above and X represents a labile group such as chlorine, bromine or iodine or a tosyloxy group or a mesyloxy group, with a cyclic amine of the formula:

H H

/ * - [s — A1 κζ \ · (III) \ / N- R --- Z 'where i z

H

20 r2, R3, R4 and n are as defined above, DK 170946 B1 6 R 'i represents a radical of the formula: - (CH2) m - CH - CH - R / 6 *' 7 5 R'6 is selected from the group consisting of of C 1 -C 14 alkylph C 1 -C 4 hydroxyalkyl, C 1 -C 4 polyhydroxyalkyl and a group of the formula:

H H

Γ "-βΓ | - \ / L. Jn \ R, - CH R- ^ I / - R .- CH /”! C »i / \ /

2 · - R, - N

2 i

H

R 2, R 3, R 4, R 7, R 11 # m and n are as defined above and Z 'is selected from oxygen and a group of the formula: \ 10 N - R'10 / R'10 is selected from the group consisting of hydrogen, C C ^alalalkyl, Cj --alk hydroxyalkyl, Cp ^ polyhydroxyalkyl and a group of the formula: hh X /

- R, 2— N - R2— V

H

R 1, R 2, R 3, R 4, R 12 and n are as defined above.

DK 170946 B1 7

The ligands of formula (I) can also be prepared according to a "StreckerM reaction by reacting a cyclic amine of formula (III) with an aldehyde of formula: R5 - CHO IIa 5 where R5 is as defined previously, in the presence of hydrogen cyanide or more common cyanide ions (KCN, NaCN).

The compounds of formula (III) wherein Z 'is a group: \ N - R10 10 / can be prepared a) by reaction of a polyamine of formula: R'

R'HN - N ^ j-R · j-NHR 'IV

n where n, R 1 and R 4 are as previously defined and R 1 represents a tosyl, mesyl or benzenesulfonyl group having a compound of the formula:

X-R, - N-R3-X V

in R 'where R 2, R 3 and R' are as previously defined and X represents a labile group such as a tosyloxy or mesyloxy group, or chloro, bromo or iodo, or b) by reacting a diamine of the formula:

R'HN - R'j - NH - R 'X

Wherein R'i and R 'are as previously defined, having a compound of the formula: Γ R'l R'

I I

5 X - R4 - N - R3 - N - R2 - X XI

n

This cyclization reaction is advantageously carried out in the presence of a phase transfer catalyst.

The polyamines of formula IV can be obtained from dihydroxylamines 10 according to the following scheme: Π Η η

HO - R4 - N - R'j - OH VI

D

4 R'Cl / pyridine Γ R'l

R'O - R4 ~ N - R'j - OR 'VII

- - * n 20 4 · NaN3 / CH3CN / H2 O R '1

N3 - R4 - N - R'j - N3 VIII

^ n

25 4 Reduction H2 Pd / C

Γ R '1

H2N - R4 - N - R'j - NH2 IX

- J n 30 4 R'Cl / pyridine DK 170946 B1 9 r R 'Ί

R'NH - R4 - N - R'j - NHR 'IV

Alternatively, phthalimide may be reacted with the compounds of formula VII and hydrazinolysis may be carried out in order to convert the compounds of formula VII into the compounds of formula IX.

The compounds of formula III, which contain two nitrogen-containing rings, can be prepared according to the methods previously described.

Thus, it is possible to react a polyamine of the formula:

/ \ XII

CH2 - CH CH - CH2 / \ I \ R'NH NHR 'R'NH NHR' wherein A and R 'are as previously defined, with a compound of formula XI to obtain a compound of formula III wherein R is a group A.

The polyamine of formula XII can be prepared from a tetrahalogenated derivative by a nucleophilic substitution in the presence of sodium azide followed by a reduction in the presence of hydrogen and palladium on charcoal.

Alternatively, those compounds of formula I, consisting of two nitrogenous rings and wherein R n is a group A, can be prepared by condensing a compound of formula:

EtOOC COOEt \ /

CH - A - CH XIII

/ \

EtOOC COOEt DK 170946 B1 10 with a polyamine of the formula: Η Η Η Γ H1

II! IN

HN - R2 - N - R3 - N - R4 - N - Η XIV

5 L Jn followed by reduction with diborane according to a method described by Tabushi et al. (Tetra Letters 12, 1049, 1977).

The compounds of formula I, which consist of two nitrogen-containing rings, are then prepared from the compounds of formula III with two rings, as previously described.

Alternatively, compounds of formula I, consisting of 2 nitrogen-containing rings, can be prepared by condensing a compound of formula I wherein R 1 is a radical of formula: 15 - (CH 2) m - CH - CH -

Re R7 wherein R $ is a hydroxyalkyl group, with an activatable bifunctional compound of the formula: 20 Xj - A - Xj

Xj is a COOH group, a COCl group or an anhydride.

The compounds of formula I, which consist of 2 nitrogen-containing rings, can also be prepared by condensing a compound of formula: DK 170946 B1 11 i5?

HOOC - CH CH - COOH

5 N- [r4 - Jl / \

R3 R "j XV

10 \ /

N- R2- N-H

HOOC - CH

R5 where R'1! is a radical of the formula: - (CH 2) m - CH - CH -

I I

R 6 is R 20 and R 7 is as previously defined and R 6 is selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 3 hydroxyalkyl and C 1 -C 6 polyhydroxyalkyl, with a compound of the formula:

X - R'12 - X XVI

X is as previously defined and R'12 represents an optionally protected R12 group.

Thus compounds of the formula are obtained: DK 170946 B1 12 r5 r5 r5 r5

H00C - CH CH - COOH HOOC - CH CH - COOH

I r h I I

5 N - R4 - N N - R4 - N

/ 1 k / \ R3 R «! R "in R3 \ / \ /

N - R2 - N - R'12 - N - R2 - N

10 I I

HOOC - CH CH - COOH

I I

%

XVII

Further, the present invention relates to complexes formed by ligands of formula I with metal ions selected from lanthanide ions having atomic numbers 57 to 71, transition metal ions having atomic numbers 21 to 29, especially Mn 2+, Fe 3+ and Cr * +, and metal ions having atomic numbers 55, 56 , 82 and 83, as well as the 20 salts of these complexes with pharmaceutically acceptable inorganic or organic bases or basic amino acids.

In such complexes, the metal ions are preferably gadolinium, europium, dysprosium, iron (Fe3 +) and manganese (Mn2 +).

Examples of salts are those formed with sodium hydroxide, N-methylglucamine, diethanolamine, lysine and arginine.

The complexes can be prepared by reacting ligands with a metal salt or metal oxide in an aqueous solvent and optionally neutralizing to form a salt.

It will be obvious that the present invention encompasses not only the ligands of formula I and the complexes previously defined in the form of racemic mixtures, but also the stereoisomeric forms of these ligands and complexes.

DK 170946 B1 13

Furthermore, the complexes of the invention may be bound to a macromolecule which may preferably be bound to an organ. Thus, the complexes of the invention can be bound to proteins and, in particular, to antibodies.

In addition, they can also be encapsulated, preferably in liposomes.

The complexes of the invention formed by the ligands of formula I with paramagnetic ions and their salts with pharmaceutically acceptable bases can be used as magnetic resonance imaging agents and as reagents which provide chemical shifts in vivo.

The complexes of the invention formed from ligands of formula I and lanthanide ions of atomic numbers 57 to 71 or metal ions of atomic numbers 55, 56, 82 and 83, and their salts with pharmaceutically acceptable bases, can be used as X-ray contrast agents.

For this purpose, especially complexes formed with the following metal ions are particularly preferred: Gd, Er, Dy, Tb, Ce, La,

Ba and Cs.

That is, the present invention also relates to diagnostic agent which can be administered to humans and comprising at least one complex formed by a ligand of formula I with metal ions selected from the lanthanide ions having atomic numbers 57 to 71, the transition metal ions having atomic numbers 21 to 29 and the metal ions having atomic numbers 55, 56, 82 and 83, as well as the salts of these complexes with pharmaceutically acceptable inorganic or organic bases or basic amino acids.

These agents may be in particular in the form of solutions of a complex of the invention in a physiologically acceptable aqueous solvent.

DK 170946 B1 14

The diagnostic agents according to the invention can be administered: parenterally, including intravenously, intra-arterially, intra-lymphatically and subcutaneously, orally, 5 - subarachnoid, - intra-bronchial in the form of an aerosol, intra-articular, - locally for visualization of cavities (eg, uterus) in magnetic resonance imaging, the doses are highly variable depending on the routes of administration.

For intravenous or intra-arterial administration, the dose is about 0.01 to 2 mM / kg.

For oral administration, this dose may be as much as 10 mM / kg.

For other routes of administration, the doses are usually less than 1 mM / kg, and for the subarachnoid route of administration it is usually even less than 0.05 mM / kg.

The doses are the same when used as reagents providing chemical shifts in vivo and as contrast agents in radiology employing x-rays, except by the intravenous or intra-arterial routes of administration, where the doses may be higher or equal to 5 mM / kg .

Furthermore, the complexes of the invention formed by the ligands of formula I and radioactive ions, as well as their salts with pharmaceutically acceptable bases, can be used as diagnostic or therapeutic agents in nuclear medicine. Examples of radioactive ions are radioisotopes of elements such as copper, cobalt, gallium, germanium, indium and, above all, technetium (Tc 99 m).

The following examples illustrate the preparation of the compounds according to the selected application.

In these examples: the NMR spectra were recorded on a 60 MHz Varian EM 360 machine with TM as internal reference. Unless otherwise indicated, the solvent is CDC13.

For example, the IR spectra were recorded on a "Perkins-Elmer 1320" device. The spectra of the solids were recorded in the form of KBr slices. In the case of liquids (oils), these were recorded in the absence of solvent.

15 - the word "buffer", used in thin layer chromatography, denotes a mixture of 1.5 M NH 4 OH and 1.5 M (NH 4) 2 CO 3.

- the melting points were measured on a "Kofler block".

the phrase used in relation to complex formation assays: "absence of free Gd3 + and of free ligands" 20 should be understood to be within the detection limits of the methods used, ie. <4 ppm and <5 ppm for Gd3 + and ligand, respectively.

Example 1 - Preparation of 2,6-dimethyl-1,4,7,10-tetraaza-cyclododecan-N.N'.N ".N" / - tetraacetic acid 25 a) Preparation of N-tosyl-bis- (2-tosyloxypropyl) amine.

A solution of 53.2 g (0.4 mole) of diisopropanolamine in 50 cm 3 of pyrldine is added dropwise while cooling to a solution of 248 g (1.3 mole) of tosyl chloride in 200 cm 3 of pyridine at 0 ° C. so that the temperature is maintained between 0 and 5 ° C. The mixture is left at this temperature for 72 hours. It is then poured into 5 2 L of water and ice and 250 cc of concentrated hydrochloric acid.

The tosyl derivative is extracted with 2 L of methylene chloride. The organic phase is dried over sodium sulfate, then filtered and decolorized with 3 SA charcoal and re-filtered through a silica layer. After evaporation of the salt, 193.8 g of a yellow oil are obtained (yield 81%, Rf = 0.7 silica / CH 2 Cl 2 / acetone / 98/2), which is used in the next step without purification.

1 HNMR spectrum: 6H CH 3 (doubled 1.2 and 1.3 ppm); 9H CH3 tosyl (singlet 2.5 ppm); 4H CH2 (multiplet centered around 3.3 ppm); 2H CH (quadrupled between 4.7 and 5.1 ppm); 12H aromatic (multiplied 7.3 and 8 ppm).

b) Preparation of N-tosyl-bis (2-azidopropyl) amine.

To 193.8 g (0.32 mol) of the compound obtained in step a) in 1.2 l of acetonitrile and 300 cm 3 of water are added 65.1 g of sodium azide (1 mol). The mixture is stirred and heated to 75 ° C for 48 hours.

After cooling, the acetonitrile is evaporated in vacuo.

The residue is taken up in 1 liter of methylene chloride. The organic phase is washed with water, dried and filtered through a silica layer (200 g). After evaporation, 82 g of a clear yellow oil is obtained (yield 75%, Rf = 0.85 silica / CH 2 Cl 2 / acetone / 92/2) which is sufficiently pure to be used directly.

IR spectrum N3 = 2100 cm -1 intense.

c) Preparation of N-tosyl-bis- (2-aminopropyl) amine.

82.0 g (0.244 mol) of the compound obtained in step b) is dissolved in 500 cm 2 of ethanol containing 8 g of 5% palladium on charcoal at 50% humidity.

The mixture was stirred vigorously while a weak stream of hydrogen was passed through (depletion of nitrogen being released). After 8 hours at ambient temperature, TLC shows that the 5 azide is not present. The mixture is then filtered and evaporated. 68.4 g of a clear yellow oil are obtained (yield 98.5%, Rf = 0.6 silica / MeOH / NH 4 OH / 95/5) which is used without purification.

NMR spectrum: δ H CH 3 (doubled 0.9 and 1 ppm); 3H CH3 tosyl (singlet at 2.4 ppm); 6H CH2 and CH (complex multiplied between 2.7 and 3.2 ppm; 4H aromatic (multiplied between 7.1 and 7.7 ppm).

d) Preparation of N-tosyl-bis [(tosylamino) propyl] amine.

93 g (0.5 mole) of tosyl chloride are added in portions to 68.4 g (0.24 mole) of the amine obtained in step c) in 500 cc of methylene chloride and 70 cc (0.5 mole) of triethylamine at 0 ° C. After the addition is complete, the mixture is stirred for 6 hours at ambient temperature. The reaction mixture is then washed with 600 cm 3 of water, the organic phase is dried, evaporated to dryness and the residue is chromatographed on a silica column with 20 pure methylene chloride and then with a methylene chloride / methanol / 98/2 mixture. The fractions of interest are evaporated and the solid residue is recrystallized from ethanol. After filtration and drying, an amount of 99.1 g (yield 70%) is obtained.

NMR spectrum: δ H CH 3 (doubled 0.9, 1 ppm); 9H CH3 p-tosyl 25 (singlet 2.4 ppm), 4H CH2 (triplet centered around 2.9 ppm); 2H CH (doubled 3.3 and 3.5 ppm); 12H aromatic (multiplied centered around 7.4 ppm).

e) Preparation of N-tosyl-bis (2-tosyloxyethyl) amine.

A solution of 32.5 g (0.31 mole) of diethanolamine in 60 cc of pyridine is slowly added to a solution of 185 g (0.97 mole) of two-column chloride in 220 cc of pyridine at 0 ° C. so that the temperature does not exceed 5 ° C. After the addition is complete, the mixture is maintained at this temperature for 1 hour and then poured into ice-cooled water (220 cc) with vigorous stirring. After filtration, washing and drying, 148.4 g of precipitate is obtained (yield 85%; Rf = 0.6 silica / CH 2 Cl 2 / acetone / 98/2).

NMR spectrum: 9H CH3 tosyl (singlet 2.4 ppm); 4H CH2N (triplet at 3.4 ppm); 4H CH2 (triplet at 4.1 ppm); 12H aromatic 10 (multiplied between 7.1, and 7.7 ppm).

f) Preparation of N, N ', N ", N"' - tetratosyl-2,6-dimethyl-1,4,7,10-tetraazacyclododecane 65 g (0.11 mol) of the compound obtained in step d), dissolved in 500 cc of dry DMF is added dropwise to 8.8 g (0.22 mol) of a 60% suspension of NaH in oil in 50 cc of DMF. The addition is carried out at ambient temperature and in such a way that there is a sustained release of hydrogen. After the addition is complete, the mixture is heated to 100 ° C and a solution of 68.1 g (0.12 mol) of the compound obtained in step e) in 20,500 cc of DMF is added dropwise. The reaction mixture is maintained at this temperature for 24 hours with vigorous stirring.

The solvent is then evaporated in vacuo and the residue taken up in a CH 2 Cl 2 / H 2 O mixture. The organic phase is washed with water, dried and evaporated to dryness. The residue (100 g) is recrystallized from isopropanol and then from toluene, and after filtration, washing with isopropyl ether and drying, 36 g of a white solid is obtained (yield 40%, Rf = 0.5-0.6 silica / CH 2 Cl 2 / acetone / 9-8 / 2).

NMR spectrum: δ H CH 3 (doubled at 1 and 1.2 ppm); 12H CH3 tosyl 30 (singlet 2.4 ppm); 14H CH2 and CH (multiplied between 3 and 4.5 ppm); 16H aromatic (multiplied between 7.1 and 7.7 ppm).

G) Preparation of N, N ', N ", N"' - tetratosyl-2,6-dimethyl-1,4,7,10-tetraazacyclododecane (variant).

A freshly prepared solution of sodium ethylate (60 mmol) in 200 cc of dry DMF is quickly added to a solution of 17 g (28.7 5 mmol) of the compound obtained in step d) in 100 cc of ethanol and refluxed. The resulting mixture becomes clear and refluxed for 1 to 2 hours. The solvents are then evaporated to dryness, the residue taken up in 200 cm 3 of DMF and heated to 100 ° C. A solution of 17 g (30 mmol) of the compound obtained in step e) in 100 cc of DMF is added to this solution over half an hour. The reaction mixture is maintained at 100 ° C overnight. The DMF is then evaporated and the residue taken up in a H 2 O / CH 2 Cl 2 mixture. The product obtained from the organic phase is chromatographed on a silica column with the mixture CH 2 Cl 2 / ethyl acetate / 98/2 as eluent. The product is recrystallized from isopropyl ether and weighed 13.5 g after drying (yield 58%, Rf = 0.5 to 0.6 silica / CH 2 Cl 2 / acetone / 98 / - 2).

Spectrum is identical to that obtained in step f).

H) Preparation of 2,6-dimethyl-1,4,7,10-tetraazacyclododecane.

33 g of the compound obtained in step f) or g) are slurried in 80 cm 3 of 98% sulfuric acid and heated to 100 ° C in an argon atmosphere for 72 hours. After being cooled, the reaction mixture is added dropwise to 1 L of ethyl ether at 0 ° C. The obtained 2,6-dimethyl-1,4,7,10-tetraazacyclododecane sulfate is filtered off, taken up in water, neutralized with sodium hydroxide and then extracted with CH 2 Cl 2. The organic phases are combined and evaporated to dryness and the resulting 6 g of solid 30 is used without further purification (yield 75%, Rf = 0.65 aluminum / butanol / water / acetic acid / 50/25 / l).

DK NMR Spectrum (D20): δH CH3 (doubled 0.9 to 1 ppm); 14 CH2 and CH (multiply centered around 2.5 ppm).

i) Preparation of 2,6-dimethyl-1,4,7,10-tetraazacyclododecane-N, N ', NM, N "' - tetraacetic acid.

A mixture of 5.7 g (60 mmol) of monochloroacetic acid and 3.4 g (60 mmol) in 25 cm 3 of water is added to a solution of 3 g (15 mmol) of the compound obtained in step h) in 25 cm 3 of water. The resulting mixture is heated to 60 ° C and a solution of potassium hydroxide (3.4 g, 60 mol) in 25 cm 3 of water is added so that the pH is maintained between 9 and 10. The addition requires 8 hours. After the addition of potassium hydroxide is complete, heating is maintained for 24 hours. After cooling, the pH is adjusted to 2.5 with concentrated hydrochloric acid. The precipitate formed is filtered off, washed with ice-cooled water and weighed 3 g after drying (yield 35%, Rf = 0.33 silica / ethyl acetate / isopropan-ol / ammonium / 12/35/30). This compound corresponds to the complex of 2,6-dimethyl-1,4,7,10-tetraazacyclododecane-N, N ', N ", - N"' - tetraacetic acid with 2 KCl.

9.5 g of this complex is eluted with 200 cc of 10% acetic acid on an ion exchange resin IRA 958 (OH), which had been previously regenerated with 1 N NaOH and washed with water until neutral. The obtained fractions were evaporated to dryness and taken up 3 times in 50 cm 3 of water to remove traces of acetic acid. The residue obtained was triturated with ethyl ether 25 (100 cm 3) and, after filtration and drying, 6.3 g of a white solid was obtained. Yield: 89%.

NMR Spectrum: (D 2 O) δ H CH 3 (doubled 1.4 and 1.5 ppm); 14H CH2 and CH (complex multiplied centered around 3.6 ppm); 8H CH2 COOH (doubled at 3.8 ppm).

DK 170946 B1 21

Example 2: Preparation of the aadolinium complex of 2,6-dimethyl-1,4,7,10-tetraazacycloododecan-N.N'.N ".N" - tetraacetic acid (methylalucamine salt)

A suspension of 5.425 g (12.54 mmol) of the 2,6-dimethyl-1,4,7,10-tetraazacyclododecane-N, N ', N ", N"' tetraacetic acid obtained in Example 1 (i) 5 and 2.27 g of Gd 2 O 3 (6.27 mmol) in 125 cm 3 of water are heated to 65 ° C for 24 hours. The pH is then adjusted to 7.4 by the addition of methylglucamine. After determining the content of free Gd3 + by the xylenol orange / EDTA method, 650 mg of 10 2,6-dimethyl-1,4,7,10-tetraazacyclododecane-N, N ', N ", N"' - tetraacetic acid (1 , 5 mmol) for complex binding of the remaining gadolinium. The completion of complex binding is determined by the absence of free Gd3 + (determined with xylenol orange) and of free ligands (complexometric determination with Cu2 +). The determination of total gadolinium in the solution is performed by atomic emission spectroscopy in DCP on a "Spectrospan 4 Beckman" apparatus. Quantitative yield, Rf = 0.49 silica / ethyl acetate / isopropanol / ammonium / 12/35/30.

Example 3; Preparation of 2-hexyl-1,4,7,10-tetraazocyclo-dodecane-N.N'.N ".N" - tetraacetic acid a) Preparation of N- (2-hydroxyethyl) -N- (2-hexyl-2 -hydroxye-thyl) amine.

50 g (0.39 mmol) of 1,2-epoxy-octane are added dropwise to 250 cm 3 (4 moles) of ethanolamine at 100 ° C. Heating is continued for 1 hour, 25 after the addition is complete. The excess ethanolamine is then distilled in vacuo. After filtration and drying, the residue is recrystallized from 600 cc of hexane. The obtained solid residue weighed 69 g (mp 45 ° C, yield = 93%, Rf = 0.62 silica / butanol / H 2 O / acetic acid / 50/25/11).

NMR spectrum: 3 H CH 3 (triplet 0.9 ppm); 10H CH2 chain (large singlet at 2.2 ppm); 7H CH2 and CH (2 poorly dissolved masses at 2.8 and 3.8 ppm).

B) Preparation of N-tosyl-N- (2-tosyloxyethyl) -N- (2-hexyl-2-tosyloxyethyl) amine.

47.3 g (0.25 mol) of the compound obtained in step a) are added in small portions to a solution of 156 g (0.82 mol) of tosyl chloride in 300 cm 3 of pyridine at 0 ° C over 1 hour. The mixture is maintained at 0 ° C for 2 days and then poured into an ice / HCl mixture (2/1). The product is extracted with CH2 Cl2 and then chromatographed on a silica column with CH2 Cl2 as eluent. The amount obtained is 118 g (yield 72%, R f = 0.6 silica / CH 2 Cl 2/10 acetone / 98/2).

NMR spectrum: 3 H CH 3 chain (tripled at 0.9 ppm); 10H CH2 chain (large singlet at 1.3 ppm); 9H CH3 tosyl (singlet at 2.4 ppm); 4H CH2N (poorly dissolved triplet at 3.4 ppm); 3H CH2O and CH (multiplied by 4.2 ppm); 12H aromatic (multiplied between 15 7 and 7.7 ppm).

c) Preparation of N-tosyl-N- (2-azidoethyl) -N- (2-hexyl-2-azodoethyl) amine.

87 g (0.133 mole) of the compound obtained in step b) and 29.25 g (0.5 mole) of sodium azide are mixed with 350 cc of acetonitrile 20 and 80 cc of water. The mixture is heated to 65 ° C for 3 days. The acetonitrile is then evaporated in vacuo and the residue taken up in CH 2 Cl 2; the organic phase is washed with water, dried and evaporated; 50 g of a yellow oil is recovered and used without purification (yield: 95%, Rf - 0.75 silica / CH 2 Cl 2 / acetone / 98/2).

25 NMR spectrum: 3 H CH 3 chain (tripled at 0.9 ppm); 10H CH2 chain (multiplied by 1.4 ppm); 3H CH3 tosyl (singlet at 2.4 ppm); 5H CH2 and CH (complex multiplied by 3.4 ppm); 4H aromatic (multiplied between 7.1 and 7.7 ppm).

IR spectrum N3 = 2100 cm -1 intense.

D) Preparation of N-tosyl-N- (2-aminoethyl) -N- (2-hexyl-2-aminoethyl) amine.

71 g (0.18 mol) of the diazide obtained in step c) is dissolved in 500 cc of ethanol to which 5 g of palladium on charcoal is added at 50% humidity. The suspension is stirred very vigorously under a hydrogen stream at ambient temperature for 24 hours. The catalyst is removed by filtration; after evaporation of the ethanol, 61.5 g of diamine is recovered, which is used without purification (yield: quantitative, Rf = 0.51 silica / MeOH: NH 4 OH / 95/5).

E) Preparation of N-tosyl-N (2-tosylaminoethyl) -N- (2-hexyl-2-tosylaminoethyl) amine.

68.6 g (0.36 mol) of tosyl chloride are added in portions to a solution of 61.5 g (0.18 mol) of the compound obtained in step d) in 500 cm 3 of CH 2 Cl 2 and 52.5 cm 3 (0.38 mol ) triethylamine at 15 ° C. After being stirred for 2 hours at ambient temperature, the reaction mixture is treated with 500 cm 3 of water. The organic phase is washed with water, dried and evaporated; the oil residue is chromatographed on a silica column with CH 2 Cl 2 as eluent. The oil obtained after evaporation of the solvent and which was taken up in isopropyl ether gives 60 g of a white solid (m.p. 120 ° C, yield 51%, Rf = 0.6 silica / CH 2 Cl 2 / MeOH / 98 / 2).

NMR spectrum: 13H hexyl chain (poorly resolved multiplet centered at 1 ppm); 9H CH3 tosyl (singlet at 2.4 ppm); 7H CH2 25 and CH (multiply centered around 3.1 ppm).

f) Preparation of N, N ', N ", N"' - tetratosyl-2-hexyl-1,4,7,10-tetraazacyclododecane.

A mixture of 46.5 g (71.5 mmol) of the compound obtained in step d) above, 41.5 g (73 mmol) of the compound obtained in Example 1 e) and 24 g (70 mmol) of tetrabutylammonium hydrogen sulfate are suspended. in 400 cc of toluene and 200 cc of 20% sodium hydroxide. The mixture is stirred very vigorously at 70 ° C for 24 hours. After cooling, the organic phase is washed with water, dried and evaporated. The residue is crystallized from ethanol and then chromatographed on a silica column with CH 2 Cl 2 as eluent. 35 g of 5 a solid is obtained (mp 154/161 ° C). Yield 56%, Rf = 0.55 silica / CH 2 Cl 2 / acetone / 98/2.

NMR spectrum: 3 H CH 3 chain (tripled at 0.9 ppm); 10H CH2 chain (multiplied by 1.3 ppm); 12H CH3 tosyl (singlet at 2.4 ppm); 15H CH2 and CH ring (multiplied by 3.3 ppm); 16H aromatic 10 (multiplied between 7.1 and 7.7 ppm).

g) Preparation of 2-hexyl-1,4,7,10-tetraazacyclododecane 12 g (13 mmol) of the compound obtained in step f) is heated to 100 ° C in 40 cm 3 of 98% sulfuric acid under argon for 24 hours. After being cooled, the mixture is added dropwise to 500 ml of ethyl ether at 0 ° C. The sulfate obtained is then filtered and neutralized with a 10% sodium hydroxide solution and extracted with CH 2 Cl 2. The organic phase is dried over sodium sulfate, then evaporated to dryness to give 2 g of a creamy solid (yield: 57%, Rf = 0.75 aluminum / butanol / -20 water / acetic acid / 50/25/11) .

The compound is stored in the form of the oxalate by reacting an ethanol-containing solution of oxalic acid with 2-hexyl-1,4,7,10-tetraazacyclododecane overnight at ambient temperature. The oxalate precipitates in the form of a white solid.

H) Preparation of 2-hexyl-1,4,7,10-tetraazacyclododecane-N, N ', N ", N"' - tetraacetic acid.

A solution of 1.09 g (2.8 mmol) of the oxalate obtained in step g) in 13 cm 3 of water and 20 ml of ethanol is neutralized with 470 mg (8.4 mmol) of potassium hydroxide. To this solution is added potassium monochloroacetic acid prepared from 1.063 g (11.25 mmol) of monochloroacetic acid and 630 mg (11.25 mmol) of potassium hydroxide in 20 cm 3 of water. The reaction mixture is heated to 60 ° C and the pH is maintained between 8 and 10 by the addition of potassium hydroxide. The addition requires 3 hours, during which time 10 cc of water containing 630 mg of potassium hydroxide is added. After a reaction time of 3 hours, 141 mg (1.5 mmol) of chloroacetic acid and 84 mg (1.5 mmol) of potassium hydroxide are added.

The mixture is maintained at 60 ° C for 2 days. After being cooled and acidified to pH 2.5 (6N HCl), the solution is passed through a column of a highly basic resin IRA 958. Elution 10 with 100 cc of 10% formic acid gives 700 mg of product. The through-flow fractions (non-retained products) are concentrated and re-treated on an identical column. After the same treatment, 2.5 g of the product was recovered (yield: "38.4%, R f = 0.65 silica / ethanol / buffer / 2/1).

NMR spectrum: 3 H CH 3 chain (triplet 0.9 ppm); 10H CH2 chain (multiplied by 1.4 ppm); 15H CH2 and CH ring (multiplied by 2.3 ppm); 8H CH2 COOH (singlet at 3.9 ppm). Spectrum recorded in D20.

Example 4: Preparation of the aadolinium complex of 2-hexyl-1,4.7,10-tetraazacyclododecane-NN '.N11 .N "-tetraacetic acid 488.6 mg (1 mmol) of the compound obtained in Example 3 and 181.3 mg (1 meq metal) gadolinium oxide is suspended in 40 cm 3 of water and heated to 65 ° C for 2 days, after 2 hours the solution is ready The progress of the complex formation is monitored during the reaction by determination of free gadolinium, and when the solution is completed, the solution is filtered through a Millipore. filter paper, then evaporated to dryness and crystallized from ethyl ether 550 mg of a white solid is thus obtained (yield: 85.5%, Rf = 0.65 EtOH / buffer / 2/1).

DK 170946 B1 26

Example 5: Preparation of the aadolinium complex of 2-hexyl 1,4,7,10-tetraazacyclododecan-NΝΝNNNN tetraacetic acid methylalucamine salt 488.6 mg (1 mmol) of the compound obtained in Example 3 3 mg (1 meq metal) of gadolinium oxide is suspended in 40 cm 3 of water and heated to 65 ° C for 12 hours. Methylglucamine is added to the clear solution to bring the pH to 7.4. Additions of ligands are made depending on the results of the assays. The completion of complexation is determined by the absence of Gd3 + (determination by xylenol orange) and by free ligands (complexometric determination with copper). The determination of total gadolinium in the solution was performed by atomic emission spectroscopy on a "Spectrospan 4 Beckmann · apparatus. Rf = 0.65 in EtOH / buffer / 2/1).

Example 6: Preparation of 2-methyl-1,4,7,10-tetraazacyclo-dodecane-N, N, N, N-tetraacetic acid a) Preparation of N, N'-ditosyl-1,2-diaminopropane.

In a 1 liter three-necked bottle equipped with a magnetic stirrer, a thermometer and a protective tube, dissolve 14.8 g of 1,2-diaminopropane in 500 ml of CH 2 Cl 2 and 58 cm 3 EfcjN.

80 g of tosyl chloride are added in portions over 1 hour. Cooling in an ice bath is necessary to maintain a temperature of 20 ° C.

The reaction mixture is then stirred overnight at room temperature.

The reaction mixture is transferred to a 1 liter separatory funnel and then washed with 2 x 260 cm 3 of water.

The organic fraction is dried over Na 2 SO 4, evaporated to dryness and then crystallized from isopropyl ether.

DK 170946 B1 27

Weight gained = 66 g Yield = 86%

Melting point = 98/100 ° C

NMR

5 1 ppm doublet (3H) 3.1 ppm multiplet (1H) 2.4 ppm singlet (6H) 5.5 ppm interchangeable singlet (2H) 2.9 ppm doublet (2H) 7.1 to 7.8 ppm aromatic ( 8H)

TLC

SiO2 eluent CH2 Cl2 90 10 MeOH, 10 Rf = 0.75.

b) Preparation of Ν, Ν'-ditosyl-bis- (2-tosyloxyethyl) ethylene diamine.

In a 500 cm 3 three-neck flask equipped with a thermometer, protective tube and magnetic stirrer, a solution of 162 g of tosyl chloride in 300 ml of pyridine is cooled to 0 ° C by an ice-salt bath.

29.6 g of this (2-hydroxyethyl) ethylenediamine is added in portions over 2 hours at this temperature. At no time should the temperature exceed 5 ° C. The reaction mixture 20 is stirred for 4 hours at this temperature, allowed to stand for 48 hours at 6/8 ° C in the refrigerator and then for 4 hours at room temperature.

The reaction mixture is poured into 1 liter of ice and water and 300 ml of concentrated HCl. The product is extracted with 500 ml of CH 2 Cl 2. The organic phase is dried over Na 2 SO 4 and then evaporated to dryness. The residue is taken up in 250 cc of ethanol under heat. The product crystallizes. It is filtered off on a glass frit and dried at 60 ° C for 48 hours.

Weight = 107.5 g 30 Yield * 70%

Melting point - 138/140 ° C

DK 170946 B1 28

NMR

2.4 ppm singlet (12H) 3.3 ppm singlet + triplet (8H) 4.2 ppm triplet (4H) 5 7.2 to 7.8 ppm multiplet (16H)

TLC

SiO2 plate, eluent toluene 80, Rf = 0.6 acetone 20 c) Preparation of N, N ', N ", NM'-tetratosyl-2-methyl-1,4,7,0ΙΟΙ tetraazacyclododecane in a 1 liter three-necked bottle a solution of 17.5 g of Ν, Ν'-ditosyldiamino-1,2-propane is stirred in 500 ml of dry DMF for one hour at ambient temperature, 33 g of Cs 2 CO 3 is pulverized and suspended in this solution. c using an oil bath in an inert atmosphere.

A solution of 35 g of Ν, Ν'-ditosyl-bis- (2-tosyloxyethyl) ethylenediamine in 200 cc of DMF is added dropwise at this temperature over 2 hours. After the addition is complete, heating is continued for 48 hours. DMF is then removed by distillation in vacuo. The residue is taken up in a water / CH 2 Cl 2 mixture.

The organic phase is dried over Na 2 SO 4. The solvent is removed by distillation in a rotary evaporator.

The residue is heated and stirred in 200 ml of ethyl acetate. The expected product crystallizes. It is filtered off and then dried at 60 ° C in vacuum for 24 hours.

Weight gained - 22.5 g Yield = 61%

Melting point = 274/275 ° C

NMR

30 1 ppm doublet (2H) 2.2 ppm singlet (12H) DK 170946 Bl 29 3 to 3.8 ppm multiplet (15H)

7.2 to 7.9 ppm multiplied (16H) aromatic TLC

SiO 2 plate, eluent toluene 80 5 acetone 20, Rf = 0.56 d) Preparation of 2-methyl-1, 4,7,10-tetraazacyclododecane In a 1 liter three-necked bottle equipped with a thermometer, an argon balloon and a mechanical stirrer are heated. a solution of 72.5 g of the compound obtained in step c) in 300 ml of 98% H 2 SO 4 10 to 100 ° C for 48 hours by means of an oil bath in an inert atmosphere.

The reaction mixture is cooled to ambient temperature and 800 ml of Et 2 O, cooled to 0 ° C by an ethylene glycol bath and dry ice, is added over 1 hour.

15 The highly hygroscopic sulfate precipitates. It is carefully filtered off on a glass frit under nitrogen and then dissolved quickly in 200 ml of water. This solution is made alkaline (NaOH beads) and then extracted with 5 x 100 ml CH 2 Cl 2.

The combined organic phases are dried over Na 2 SO 4, then evaporated to dryness to give 15 g of a highly viscous crude product which crystallizes on standing.

NMR

CDCl3 1.1 ppm doublet (2H)

Spectrum in + D20 2.7 ppm 2 singlets 25 as a multiplet (19H, 4 which are interchangeable)

TLC

A1203 plate, eluent BuOH 50, Rf 0.8 0.8 H2 O AcOH 11 e) Preparation of the complex of 2-methyl-1,4,7,10-tetraazacyclododecane-Ν, Ν ', NM, N "' - tetraacetic acid with 2 KC1.

In a 250 ml three-necked bottle, 34 g of chloroacetic acid in 150 cm 3 of water are cooled to 10 ° C in an ice bath. 20 g of potassium hydroxide are added at this temperature to neutralize the acid.

The compound obtained in step d) is then dissolved in this solution. The reaction mixture is heated to 65 ° C by means of an oil bath.

A solution of 20 g of KOH in 50 cm 3 of water is gently added over 10 hours at this temperature while maintaining the pH between 8 and 10.

Heating is then continued for 72 hours, after which the reaction mixture is acidified to pH = 2.5 with concentrated HCl.

The complex precipitates. It is filtered off on a glass fryer, purified with 50 cm 3 of water and then dried at 60 ° C for 18 hours in an oven.

Weight gained = 30 g Yield = 66%

Melting point> 300 ° C

F) Purification of 2-methyl-1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid 30 g of the complex obtained in step e) is suspended in the presence of 150 cc of IRA 958 resin, as previously described. has been regenerated.

After dissolving the complex, this suspension is loaded onto a column containing 150 cc of IRA, 958 resin.

DK 170946 B1 31

The elution is performed with a 5% acetic acid solution in water.

The fractions containing the expected pure product are evaporated to dryness to completely remove the acetic acid.

5 Weight gained = 18.4 g Yield = 89%

Acidity = 100.3% (4 equivalents) titrated with 0.1 M

NaOH.

TLC

10 SiO2 eluent AcOEt 12, Rf * 0.36 isopropanol 35 NH3, H2 O FAB mass spectrum: mass peak at M + 1 * 419.

Example 7; Preparation of a solution of the aadolinium-15 complex of 2-methyl-1,4,7,10-tetraazacycloododecane-NN, N11.N, t and 9.05 g (25 mmol) of gadolinium oxide are dissolved in 50 ml of double-distilled deaerated water at 70 ° C. After 1 hour, the solution 20 is completed and the pH is close to 3. After cooling, the pH is adjusted to 7.3 with methylglucamine. The solution is filled to 100 ml and filtered through a 0.22 mm Millipore membrane. A solution having a Gd content of 0.5 mol / l is thus obtained. This solution has a viscosity at 20 ° C of less than 4 mPa.s (free Gd not 25 trace).

Example 8; 2-Hydroxymethyl-1,4,7,10-tetraazacyclododecane-4.7.10-acetic acid a) Preparation of N, N'-dithosyl-2,3-diaminopropionic acid.

To a solution of 46 g of sodium hydroxide in 500 cm 3 of water is added 40 g of the monohydrochloride of 2,3-diaminopropionic acid with vigorous stirring. Ethyl ether (200 cc) followed by 110.5 g of two-chloride chloride is added portionwise over 1 hour. Stirring is maintained for 12 hours, the precipitate formed is filtered from 5 and then washed with water and ethyl ether. The obtained solid is suspended in ill water and then acidified with 6 N HCl. After filtration and washing with water and ethyl ether, the solid is dried for 24 hours at 60 ° C in vacuo.

Weight gained = 76 g 10 Yield - 65%

Melting point = 200-201 ° C

TLC: SiO 2 CH 2 Cl 2 80 / MeOH 20

Rf = 0.5

NMR

15 - 2.4 ppm singlet 6H (CH3 from the tosyl group) - 2.8 ppm multiplet 3H (CH2, CH from the diamino chain) - 3.5 to 5 ppm extended multiple 3H interchangeable with D20 - 7.2 to 7.8 ppm multiplet 8H aromatic.

b) Preparation of N, N / -ditosyl-2,3-diaminopropanol 20 In a 2 liter three-neck flask, a suspension of 40 g of the compound obtained in step b) is stirred in 600 cm 3 of THF at 20 ° C in an inert and anhydrous atmosphere (argon).

A solution of 500 ml of 1M BH3: THF is added in an inert atmosphere over half an hour. The temperature of the reaction mixture rises to 30 ° C. Stirring is continued for 48 hours. Hydrolysis is performed gently with 20 ml of water. THF is removed by distillation in vacuo. The residue is extracted with a water / ether mixture. The organic phase is washed with water, dried over Na 2 SO 4 and then evaporated to dryness. The residue is triturated with isopropyl ether until crystallized. After filtration and drying, 35 g of the product is obtained.

DK 170946 B1 33

Yield: 90%

Melting point: 126-127 ° C TLC: SiO2 CH2 Cl2 90 / MeOH Rf: 0.6

NMR

- 2.7 ppm singlet "CH3" from tosyl (6H) - 3 to 3.7 ppm multiplet (7H, 2 which are interchangeable) - 6.9 ppm triplet, interchangeable alcoholic OH (1H) - 7.3 to 7, 9 ppm Multiple Aromatic (8H) 10 c) Preparation of N, N ', N ", N"' - tetratosyl-2-hydroxymethyl-1,4,7,10-tetraazacyclododecane.

In a 2 liter three-necked flask under argon, 35 g of the compound obtained in step b) is dissolved in anhydrous DMF and then 58.6 g of anhydrous Cs 2 CO 3 is added.

This suspension is stirred for 1 hour at ambient temperature then heated to 65 ° C by means of an oil bath. A solution containing 69 g of N, N'-ditosyl-bis- (2-tosyloxyethyl) -ethylenediamine in 600 cc of anhydrous DMF is added dropwise at this temperature over 6 hours. Heating to 65 ° C is maintained overnight; DMF is removed by distillation in vacuo. The residue is taken up in a mixture of 400 ml of water and 400 cm 3 of dichloromethane. The organic phase is decanted, washed with 200 cm 3 of water, dried over Na 2 SO 4 and then evaporated to dryness. The residual oil is dissolved at 80 ° C in 200 cc of toluene, then left in a refrigerator for 48 hours, allowing crystallization to take place. 24 g of the product is obtained.

Yield: 32%

Melting point: 143-145 ° C TLC: SiO2 CH2 Cl2 90 / AcOEt Rf: 0.5 DK 170946 B1 34

NMR

2.4 ppm singlet 12H CH3 tosyl

3.2 to 4.1 ppm multiplied by 17 CH2 ring + CH2-0H

7.2 to 8.1 ppm multiplied 16H aromatic 5 d) Preparation of 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane.

20 g of the compound obtained in step c) are dissolved in 100 cm 3 of 98% H 2 SO 4. The solution is heated to 100 ° C for 48 hours in an inert atmosphere. The reaction mixture is cooled and then added dropwise to 1 L of ethyl ether which is cooled by a dry ice / acetone bath.

The precipitate of the amine sulfate is filtered off on a glass frit and then washed with ethyl ether. The solid is immediately dissolved in 200 cm 3 of water, adjusted to pH 12 with NaOH and evaporated to dryness. After drying the residual solid in vacuo in the presence of P 2 O 5, the product is extracted by refluxing with 2 x 100 cm 3 of THF. Evaporation of the fractions obtained after extraction leads to a colorless oil.

20 Weight obtained: 4.5 g base Yield: 90% TLC: Al2 O3 BuOH 50 / water 25 / AcOH 11 Rf: 0.8 NMR (CDCl3 spectrum) 25 2.8 ppm singlet (17H) + triplet 3.8 ppm exchangeable singlet (5H) e) Preparation of 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid.

In a 250 cm 3 three-neck flask equipped with a heat-magnetic stirrer, a temperature probe and a pH electrode connected to an analog pH meter, and a reagent addition system, correlated with the pH of the medium , a solution of 8.5 g of the compound obtained in step d), 15.8 g of 2-chloroacetic acid and 100 ml of water is neutralized to pH = 9.5 by means of a solution of 15.8 g of potassium hydroxide in 50 cm 3 water. Then, the reaction mixture is heated to 50 ° C on an oil bath for 72 hours. Further, the pH of 9.8 is maintained by continuous addition of a potassium hydroxide solution. The mixture is cooled, acidified to pH = 5, diluted to 500 cm 3 and applied to a 500 cm 3 column of the highly basic anion exchange resin IRA 958, which has been previously regenerated. The alkylation products bind to the resin. The latter is purified with water and then eluted with 5% acetic acid fractions. The fractions are evaporated to dryness. The residue is a crude powder which is purified on a preparative HPLC column of diameter 40, covered with RP.18 grafted silica.

Weight achieved: 3.5 g of pure product Yield: 22%

Melting point: 142-144 ° C

TLC: SiO 2, AcOEt 12 / isopropanol 35 / NH 4 OH Rf: 0.35

Acidity: 198.7% (2 waves)

Titration with 0.1 M NaOH - corrected for H2 O FAB mass spectrum: peak at M + 1 = 377

Example 9: Preparation of a solution of the cradolinium complex of 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane 4.7.10-acetic acid.

A suspension of 11.05 g of 2-hydroxymethyl-1,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid and 5.07 g of gadolinium oxide in double-distilled water is heated to 80 ° C for 1 hour.

After cooling, the pH is adjusted to 7.3 by the addition of sodium hydroxide and the volume is adjusted to 100 ml. Determination of total gadolinium is performed by atomic emission spectroscopy (0.2 DK 170946 B1 36

Ml-1).

Example 10: Preparation of 2-hydroxymethyl-1,4,7,10-tetrazacycloododecane-1.4.7.10-tetraacetic acid.

In a 50 cm 3 reactor equipped with a magnetic stirrer, a solution of 0.7 g of 2-hydroxymethyl-1, 4,7,10-tetra-azacyclododecane-4,7,10-triacetic acid and 0.2 g is heated. g of chloroacetic acid in 15 cm 3 of water to 70 ° C.

The pH is adjusted to 10.5 by a solution of potassium hydroxide and maintained at this value for 48 hours at 70 ° C.

When the reaction is complete, the pH is adjusted to 5 and then the solution is applied to an IRA 958 resin.

The ligand is chromatographed on the resin by elution with 5% acetic acid.

After evaporation to dryness, the product is purified by preparative HPLC (RP18 - grafted silica).

Thus, 0.15 g of ligand is recovered in a yield of 18%.

TLC (silica), eluent ethyl acetate 12 isopropanol 35, R f = 0.25 NH 3, H2 O FAB mass spectrum: Peak at M + 1 = 435.

Example 11: Preparation of 2- (2-hydroxymethyl) -1.4.7.10-tetrazaacycloododecane-N.N '.NH .N' - tetraacetic acid.

This ligand is prepared according to the method of synthesis of 2-hydroxymethyl-1,4,7,10-tetra-azacyclododecane-Ν, Ν ', NH, N "-tetraacetic acid described in Examples 8 and 10, starting from 3, 4-diaminobutanoic acid (S. Kasina et al., J. Med. Chem., 29, 1933, 1986).

DK 170946 B1 37

Example 12; Preparation of 2-methyl-1,4.7.10.13-pentaaza-cyclopentadecane-4.7.10.13-tetraacetic acid in product 12a) and 2-methyl-1,4.7.10.13-pentaazacyclopentadecane-1.4.7.10.13-penta-acetic acid (product 12b ^ .

A) Preparation of 1,4,7,10,13-pentatosyl-2-methyl-1,4,7,10,13-pentaazacyclopentadecane.

35.7 g (0.093 mole) of N, N'-ditosyl-1,2-diaminopropane, 75.7 g of cesium carbonate (0.23 mole) and 800 ml of DMF are poured into a 2 L three-necked flask equipped with a refrigerant and a mechanical stirrer.

The mixture is brought under argon and heated to 75 ° C.

A solution of 83 g (0.012 mol) of 1,11-mesyloxy-3,6,9-tritosyl-3,6,9-triazaundecane synthesized according to Richman and Atkins, Organic Synthesis 58, page 86, in 700 ml of DMF is added to -15 over 4 hours at 75 ° C.

The reaction mixture is maintained at 75 ° C for 48 hours, then filtered and the filtrate is evaporated to dryness.

The residue is taken up in 700 ml of ethanol, the solid is filtered off and then taken up in 800 ml of toluene under heating.

The solid is filtered off at room temperature and then dried at 60 ° C.

Weight achieved: 45.4 g Yield: 49%

Analysis: TLC: SiO2 60 F 254 Merck Eluent CH2 Cl2 / acetone 98.2, Rf 0.45

Mass Spectrum DCI Method (NH3)

Mass peak at 999.

B) Preparation of 2-methyl-1,4,7,10,13-pentaazacyclopenta-decane.

44 g (0.044 mol) of the compound obtained in step a) are maintained for 72 hours at 100 ° C in 130 ml of concentrated sulfuric acid.

After being cooled, the reaction mixture is poured into a mixture of 250 ml of ethyl ether and 250 ml of ethanol at 0 ° C.

The solid is filtered off, then dissolved in 250 ml of water and treated with carbon black. The solution is made alkaline with cesium hydroxide and then extracted with CH 2 Cl 2. The organic solution is dried over Na 2 SO 4 and evaporated to dryness. The amine thus obtained can be used as such or in the form of its hydrochloride.

Weight obtained: M = 8.4 g as the free base M = 13.2 g as 5 HCl Yield: 72.8%

Analysis of the hydrochloride: TLC: A1203 F 254 Merck Eluent: ethanol / isopropylamine 80/20 Developer: iodine, Rf: 0.85 NMR: 1.7 ppm 3H CH3 3.7 ppm 19H CH2 and CH.

c) Preparation of 2-methyl-1,4,7,10,13-pentaazacyclopentadecane-4,7,10,13-tetraacetic acid (product 12a) and 2-methyl-1,4,7,10,13- pentaazacyclopentadecan-1,4,7,10,13-pentaacetic acid (product 12b).

In a 500 ml three-neck flask, a solution of 25.7 g (0.27 mole) of chloroacetic acid in 50 ml of water is neutralized to pH * 5 at T <10 ° C using 5 M potassium hydroxide.

10 g of the compound (10.043 mmol) obtained in step b) are dissolved in 20 ml of water and added to this solution. The mixture DK 170946 B1 39 is heated to 55 ° C and 50 ml of 5 M potassium hydroxide is added over 48 hours to maintain the pH of 8.5-9.5. After the addition is complete, heating is continued overnight. The reaction mixture is cooled and acidified to pH = 3. The solution is then added to 200 ml of DOWEX 50 W resin. Elution of the resin by means of 1 1 1 M aramonium solution leads to the recovery of 20 g of crude product. The crude product is dissolved in 150 ml of water and 250 ml of IRA 958 resin is applied. The resin is washed with water, then eluted with 2 L 0.1 M acetic acid followed by 2 L 0.8 M acetic acid.

9 g of crude product 12a is obtained by evaporation of the 0.1 M acetic acid. 2.5 g of crude product 12b is obtained by evaporation of the 0.8 M acetic acid.

Products 12a and 12b are then purified by preparative HPLC on 15 RP18 silica.

Product obtained: product 12a: M = 5 g product 12b: M = 1.1 g

Yield: 30%

Analysis: TLC: si02 60 F 254 Merck

Eluent: ethyl acetate / isopropanol / NH 3 (30%) / 12/35/30 Developer: Iodine Product 12a, Rf: 0.4 Product 12b, Rf: 0.27 Water Analysis:

Product 12a: KF: 1.8%

Product 12b: KF: 2.8%

Acidity analysis using 0.1 M NaOH:

Product 12a: 2 acidity analyzes 98.6% and 100.6%, titers: 99.6% Product 12b: 3 acidity analyzes 199.2% and 92.4%, titers: 97.3% FAB mass spectra: 12a peak at M + 1 462 12b top at M + 1 520 DK 170946 B1 40

Comparative Example 13 This example illustrates a comparison experiment that has been made between the nearest known complex Gd-DOTA and Gd-MCTA corresponding to the metal complex of Example 7 in the present text.

The structural formulas for DOTA and MCTA respectively are as follows: ch3

HOOC — v / \ s — COOH HOOC — v f ~ \, —COOH

NN N N

(

NN NN

HOOC— / ^ —COOH HOOC— ^ ^ '- COOH

DOTA MCTA

In the experiment, the LD50 value (i.e., the dose that induces death in 50% of a population) was determined by intravenous administration to mice and dissociation constants for the two complexes. The results are given in Table 1 below.

TABLE 1

Connections Gd-MCTA Gd-DOTA

LD50 IV (mouse 15.37 11.5 (mmol / kg))

Dissociation 2.7 · 10 * 9 + 4.5 10'7 (H +) 6 · 10'9 + 8 · 10'7 (H +) constant at pH> 6 at pH> 6 K half-life> 8 years half-life> 3 year

As can be seen from the table, the Gd-MCTA of the invention has 20 lower dissociation constant and lower toxicity than the nearest known unsubstituted homologous Gd-DOTA.

Claims (20)

1. Ligand, characterized in that it has the formula: R, R5 in 3 in 3 H00 C - CH CH COOH N - [n] / "\ R, R, 1 \ / N N- R, -ZI £ H00C- CH R5 where 2HH R2, R3, R4, R7, Rn, m and n are as defined above and Z 'is selected from the group consisting of an oxygen atom and a group of the formula: 10 \ N - R'10 / R'10 is selected from the group consisting of hydrogen, Cj-C ^ alkyl, Cj-C4 hydroxyalkyl, CX-C ^ polyhydroxyalkyl and a group 15 of the formula: HH \ / R -, R₂, N— R₂, RH R₂, R₂ , R3, R4, R12 and n are as defined above. DK 170946 B1 2. R, - N Compound according to claim 1, characterized in that it is 2,6-dimethyl-1,4,7,10-tetraazacyclododecane-N, N / - 3 R · Irr \ / 'N-R - £ ·. In 24 H where R2, R3, R4 and n are as defined above, R'j represents a radical of the formula: DK 170946 B1 “(CH2) m - CH - CH - R / 6 R? R 6 is selected from the group consisting of C 1 -C 14 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 polyhydroxyalkyl and a group of the formula: H H ΓΑ -., Μ. n / J- \ R, -CH R / <CH) / 'X / A compound according to claim 1, characterized in that it is 2-hexyl-1,4,7,10-tetraazacyclodecane-N, N ', N ", N"' - tert-acetic acid. A compound according to claim 1, characterized in that it is 2-methyl-1,4,7,10-tetraazacyclododecane-N, N ', N ", - N"' - tetraacetic acid. DK 170946 B1 Compound according to claim 1, characterized in that it is 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid. C C-C ^ hydroxyalkyl, C C-C C polyhydroxyalkyl, a group of the formula -CH - COOH wherein R 5 is as defined previously, and a * 5 group of the formula: I * 5 A 'C H-COOH CH-COOH / - '·! - \ \ /' - R - N - - N 12. i CH-C00H * 5 R 1 represents a radical of the formula: - (CH 2) m - CH - CH - II * 6 * 7 is selected from the group consisting of C 1 -C 14 alkyl, C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl and a group of the formula: r RI 3 I 5 CH-COOH CH-COOH p -% - \ R7_CH (CH) / 'X / Z - R, - N 2 in CH-COOH R5 DK 170946 B1 Ru is selected from the group consisting of groups A and the groups of the formula: ~ (ch 2) t - y - a - y - (CH 2) t - A are selected from the group consisting of C 1 -C 8 alkylene, C 1 Y is selected from -CO- and - O and t = 1 to 4. o Compound according to claim 1, characterized in that it is 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane-1,4- 7,10-tetraacetic acid. A compound according to claim 1, characterized in that it is 2-hydroxyethyl-1,4,7,10-tetraazacyclododecan-1,4,4,7,10-tetraacetic acid. Process for the preparation of a compound according to claim 1, characterized in that it comprises the reaction of a compound of the formula: X - CH - COOH II A process according to claim S, characterized in that it comprises the preparation of the compound of formula III by reacting a polyamine of the formula: 'r * R'H N _ [' _ νΊ_R '1 -NHR * IV 5 wherein R R 2 is as defined in claim 8, and R 'is a tosyl, roesyl or benzenesulfonyl group, having a compound of the formula: X-R4-N-R3-X V Process according to claim 8, characterized in that it comprises the preparation of the compound of the formula R 'wherein R 3, R 4 and R' are as defined in claim 8 and X represents a labile group. R 12 is selected from the group consisting of C 1 -C 8 alkylene, C 1 -C 8 hydroxyalkylene and C 1 -C 8 polyhydroxyalkylene, as well as salts thereof. R 7 is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl, m = 0 or 1, R 2, R 3, R 4 are the same or different and represent a radical of the formula : 15 - (CH) _ - CH - II R 9 R 9 R 9 and R 9 are the same or different and are selected from hydrogen, C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl, 20. are 1 or 2 , n is 0, 1 or 2 and R 5 is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl, and Z is selected from the group consisting of oxygen and a group of the formula : \ DK 170946 B1 N - R10 / R10 is selected from the group consisting of hydrogen, C1-C4 alkyl, Complexes formed from ligands of formula I according to claim 1, characterized in that it is the complex of ligand DK 170946 B1 and metal ions selected from the group consisting of lanthanide ions (atomic numbers 57 to 71), transition metal ions (atomic numbers 21 to 29) and metal ions having atomic numbers 55, 56, 82 and 83, as well as the salts of these complexes with pharmaceutically acceptable inorganic or organic bases or with basic amino acids. Complexes according to claim 11, characterized in that the metal ion is selected from the group consisting of gadolinium, europium, dysprosium, iron (Fe3 +) and manganese (Mn2 +). Complex according to claim 12, characterized in that it is the methylglucamine salt of the gadolinium complex of 2,6-dimethyl-1,4,7,10-tetraazacyclododecane-N, N ', N ", N"' - tetraacetic acid. Complex according to claim 12, characterized in that it is the methylglucamine salt of the gadolinium complex of 2-hexyl-1,4,7,10-tetraazacyclododecane-N, N ', NH, N "-tetraacetic acid. Complex according to claim 12, characterized in that it is the methylglucamine salt of the gadolinium complex of 2-methyl-1,4,7,10-tetraazacyclododecan-Ν, Ν ', N ", N"' - tetraacetic acid. III by reacting a diamine of the formula: R'HN - R'j - NH - R 'X wherein R'j and R' are as defined in claim 8, with a compound of the formula: Γ R1 R / 20. In X - R4 - N - R3 - N - R2 - X XI D wherein n, R2, R3, R4 and R 'are as defined in claim 8 and X represents a labile group. R5 wherein R5 is as defined in claim 1 and X represents a labile group or an aldehyde of the formula: R5 - CHO IIa wherein R5 is as defined in claim 1, in the presence of hydrogen cyanide or cyanide ions, with a cyclic amine with the formula: "- HP" N, NM'-tetraacetic acid. Complex according to claim 12, characterized in that it is the gadolinium complex of 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid. Diagnostic agent which can be administered to humans, characterized in that it comprises at least one complex according to any one of claims 11-16. Agent according to claim 17, characterized in that it consists of a solution of the complex in an aqueous solvent. DK 170946 B1 A magnetic resonance imaging agent, characterized in that it comprises at least one complex formed by a ligand according to claim 1, with paramagnetic metal ions, and salts thereof with pharmaceutically acceptable bases. 5 X-ray contrast agent, characterized in that it comprises at least one complex formed by a ligand according to claim 1, with lanthanide ions having atomic numbers 57 to 71, and metal ions having atomic numbers 55, 56, 82 and 83 and salts thereof having pharmaceutically acceptable bases . 10